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Response of the Nervous System 
to Ionizing Radiation 



Response of the Nervous System 
to Ionizing Radiation 



Proceedings of an International Symposium 

held at Northwestern University Medical School 

Chicago, Illinois, September 7-9, 1960 



Edited by 
THOMAS J. HALEY 

Laboratory of Nuclear Medicine 

and Radiation Biology 

School of Medicine 

University of California Medical Center 

Los Angeles, California 



RAY S. SNIDER 

Northwestern University Medical School 
Chicago, Illinois 



Assistant Editor 

SHIRLEY MOTTER LINDE 
1962 





ACADEIVIIC PRESS - New York and London 



Copyright © 1962, by Academic Press Inc. 



ALL RIGHTS RESERVED 



NO PART OF THIS BOOK MAY BE REPRODUCED IN ANY FORM 

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United Kingdom Edition 
Published by 

ACADEMIC PRESS INC. (London) Ltd. 

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Library of Congress Catalog Card Number 61-18880 



s ^ 




printed in the united STATES OF AMERICA 



CONTRiBUTORS 

Bengt Andersson, Gustaf Werner Institute for Nuclear Chemistry and the 

Institute of Anatomy, University of Uppsala, Uppsala, Sweden 
William J. Arnold, University of Nebraska, Lincoln, Nebraska 
George Austin, University of Oregon Medical School, Portland, Oregon 
C. S. Bachofer, University of Notre Dame, Notre Dame, Indiana 
Orville T. Bailey, University of Illinois College of Medicine, Chicago, 

Illinois 
Charles P. Baker, Brookhaven National Laboratory, Upton, New York 
John S. Barlow, Massachusetts General Hospital, Boston, Massachusetts 
Albert Behar, Armed Forces Institute of Pathology, Washington, D. C. 
Leslie R. Bennett, University of California, Los Angeles Medical Center, 

Los Angeles, California 
A. Breit, Deutsche Forschungsanstalt fiir Psychiatric, Max-Planck-Institut, 

Munich, Germany 
K. R. Brizzee, University of Utah College of Medicine, Salt Lake City, Utah 
Daniel G. Brown, University of Tennessee — Atomic Energy Commission, 

Agricultural Research Laboratory, Oak Ridge, Tennessee 
VV. Lynn Brown, University of Texas, Austin, Texas 

Robert H. Brownson, Medical College of Virginia, Richmond, Virginia 
Tor Brustad, Norwegian Radium Hospital, Oslo, Norway 
W. G. Calvo, Brookhaven National Laboratory, Upton, New York 
Berry Campbell, College of Medical Evangelists, Los Angeles, California 
A. Carsten, Brookhaven National Laboratory, Upton, New York 
Carmine D. Clemente, University of California, Los Angeles Medical 

Center, Los Angeles, California 
Howard J. Curtis, Brookhaven National Laboratory, U pton. New York 
Roger T. Davis, University of South Dakota, Vermillion, South Dakota 
Donald Duncan, University of Texas Medical Branch, Galveston, Texas 
L. E. Farr, Brookhaven National Laboratory, Upton, New York 
C. T. Gaffey, Donner Laboratory, University of California, Berkeley, 

California 
Edgar L. Gasteiger, University of Rochester, School of Medicine and Den- 
tistry, Rochester, New York 
N. I. Gr,\shchenkov, Institute of the Higher Nervous Activity, U.S.S.R. 

Academy of Sciences, Moscow, U.S.S.R. 
H. Hager, Deutsche Forschungsanstalt fiir Psychiatric, Max-Planck-Institut, 

Germany 
Harry F. Harlow, University of Wisconsin, Madison, Wisconsin 
W. Haymaker, Brookhaven National Laboratory, Upton, New York 



vi CONTRIBUTORS 

Julian Henry, Donner Laboratory, University of California, Berkeley, 

California 
Paul S. Henshaw, Division of Biology and Medicine, Atomic Energy Com- 
mission, Washington, D. C. 
Samuel P. Hicks, Harvard Medical School and New England Deaconess 

Hospital, Boston, Massachusetts 
Wolfgang Hirschberger, German Research Institute of Psychiatry, Max- 

Planck-Institute, Munich, Germany 
J. R. M. Innes, Brookhaven National Laboratory, Upton, New York 
Walter Isaac, University of Washington School of Medicine, Seattle, 

Washington* 
L. A. Jacobs, University of Utah College of Medicine, Salt Lake City, Utah 
Peter Janssen, Donner Laboratory, University of California, Berkeley, 

California and Armed Forces Institute of Pathology, Washington, D. C.f 
Larry P. Jones, University of Tennessee — Atomic Energy Commission, 

Agricultural Research Laboratory, Oak Ridge, Tennessee 
Sylvan J. Kaplan, Texas Technological College, Lubbock, Texas 
X. Kharetchko, University of Utah College of Medicine, Salt Lake City, 

Utah 
Donald J. Kimeldorf, U. S. Naval Radiological Defense Laboratory, San 

Francisco, California 
Igor Klatzo, National Institute of Neurological Diseases and Blindness, 

National Institutes of Health, Bethesda, Maryland 
Ruth Kleinfeld, Ohio State University, Columbus, Ohio 
Harold Koenig, Veterans Administration Research Hospital and North- 
western University Medical School, Chicago, Illinois 
Lawrence Kruger, University of California Medical Center, Los Angeles, 

California 
P. Krupp, University of Basel, Basel, Switzerland 
BoRjE Larsson, Gustaf Werner Institute for Nuclear Chemistry and the 

Institute of Anatomy, University of Uppsala, Sweden 
Robert W. Leary, University of Washington School of Medicine, Seattle, 

Washington^ 
Lars Leksell, Gustaf Werner Institute for Nuclear Chemistry and the 

Institute of Anatomy, University of Uppsala, Sweden 
Billey Levinson, University of Buffalo, Buffalo, New York 
Leo E. Lipetz, Institute for Research in Vision, Ohio State University, 

Columbus, Ohio 
S. W. LipPiNCOTT, Brookhaven National Laboratory, Upton, New York 

* Present address: Emory University, Atlanta, Georgia 
t Present address: Institut Neurologique Beige, Brussels, Belgium 
Present address: University of Oregon, Eugene, Oregon 



CONTRIBUTORS vii 

John Lvman, Donncr Laboratory, University of California, Berkeley, 

California 
Arnold A. McDowell, University of Texas, Austin, Texas 
William Mair, Gustaf Werner Institute for Nuclear Chemistry and the 

Institute of Anatomy, University of Uppsala, Uppsala, Sweden 
Leonard I. Malis, Mt. Sinai Hospital, New York, New York 
Dorothea Starbuck Miller, University of Chicago, Chicago, Illinois 
Jaime Miquel, National histitute of Neurological Diseases and Blindness, 

National Institutes of Health, Bethesda, Maryland 
Marcel Monnier, University of Basel, Basel, Switzerland 
Werner K. Noell, Roswell Park Memorial Institute, Buffalo, New York* 
Nancy Ragan, University of California School of Medicine, Los Angeles, 

California 
Bror Rexed, Gustaf Werner Institute for Nuclear Chemistry and the In- 
stitute of Anatomy, University of Uppsala, Uppsala, Sweden 
Harold E. Richardson, Jr., Donner Laboratory, University of California, 

Berkeley, California 
Arthur J. Riopelle, Yerkes Laboratories of Primate Biology, Orange Park, 

Florida 
Jerzy E. Rose, The University of Wisconsin, Madison, Wisconsin 
T. G. Ruch, University of Washington School of Medicine, Seattle, 

Washington 
Roberts Rugh, Radiological Research Laboratory, Columbia University, 

New York, New York 
Daniel P. Sasmore, University of Tennessee — Atomic Energy Commission, 

Agricultural Research Laboratory, Oak Ridge, Tennessee 
Makoto Sato, University of Oregon Medical School, Portland, Oregon 
Mary Elmore Sauer, University of Texas Medical Branch, Galveston, 

Texas 
Dante G. Scarpelli, Department of Pathology, The Ohio State University, 

Columbus, Ohio 
O. A. Schjeide, University of California School of Medicine, Los Angeles, 

California 
Wolfgang Schlote, German Research Institute of Psychiatry, Max- 

Planck-Institute , Munich, Germany 
WiLLiBALD ScHOLZ, German Research Institute of Psychiatry, Max-Planck- 

Institute, Munich, Germany 
Norbert Schummelfeder, histitute of Pathology, University of Bonn, 

Germany 
J. G. Sharp, University of Utah College of Medicine, Salt Lake City, Utah 

•Present address: University of Buffalo Medical School, Buffalo, New York 



viii CONTRIBUTORS 

Sue Simons, University of California School of Medicine, Los Angeles, 

California 
Patrick Sourander, Gustaf Werner Institute for Nuclear Chemistry and 

the Institute of Anatomy, University of Uppsala, Uppsala, Sweden 
Walter R. Stahl, Oregon State College, Corvallis, Oregon and University 

of Oregon Medical School, Portland, Oregon 

E. E. StickleYj Brookhaven National Laboratory, Upton, New York 
Cornelius A. Tobias, Donner Laboratory, University of California, Berke- 
ley, California 

D. C. Van Dyke, Donner Laboratory, University of California, Berkeley, 
California 

F. Stephen Vogel, New York Hospital, Cornell University Medical Cen- 

ter, New York, New York 
Y. L. Yamamoto, Brookhaven National Laboratory, Upton, New York 
James N. Yamazaki, University of California, Los Angeles Medical Center, 

Los Angeles, California 
Wolfgang Zeman, Indiana University Medical School, Indianapolis, 

Indiana 



FOREWORD 



The assembly of groups of people of divergent views for the purpose 
of educating each other is a goal which is not too often attained. However, 
these directions were the ones given to the Symposium and program chair- 
men by the committee that had decided it was time to look into effects of 
ionizing radiations on the nervous system. 

This was a new approach, because many had said that the nervous 
system was insensitive to radiation, but the undercurrent arriving from 
many laboratories indicated that the statement was only partly true. If we 
wished to understand the radiation syndrome itself it would be necessary 
to consider the ner\ous system in our o\er-all outlook. Upon this basis it 
was decided that a beginning should be made by reviewing neonatal 
aspects, histopathological effects, ablation of specific central nervous system 
areas by particulate irradiation, ev^aluation of functional changes, and last 
but not least, the psychological effects of irradiation on animal performance. 

To further these goals, investigators from many parts of the world 
joined with their colleagues in the United States to present the material 
contained in the following pages. We do not believe that all available 
information on the subject is contained herein, but we have strived honestly 
for a beginning in order that investigators will not only know what has been 
done and is being done, but also what the future may cause to have done. 

This symposium was made possible by research grants to Northwestern 
University from the Institute of Neurology and Blindness, National Insti- 
tutes of Health, Bethesda, Maryland, and the U. S. Atomic Energy Com- 
mission, Washington, D. C. Special thanks go to the Neurology Study Sec- 
tion of National Institutes of Health for the necessary services it rendered. 

We hope that this modest beginning will inspire others to assist all to 
a better understanding of the various effects produced by irradiation of 
the nervous system. 

Thomas J. Haley 
Program Chairman 
Ray S. Snider 
Symposium Chairman 



PREFACE 



This is a meeting of two groups of minds, the basic neurologists and 
the basic radiobiologists. It is the first meeting of its kind and, if successful, 
we hope there will be subsequent meetings. It should not be necessary to 
point out that this select group has a double responsibility, that of pointing 
up not only what we know, but, equally important, what we don't know 
about radiation effects on the nervous system. This is a long neglected field, 
and we are all students with much to learn. On some of the points there is 
enough information for general agreement; on other points, general agree- 
ment is impossible. Perhaps the frustrated feelings will be so annoying that 
you will go back to your laboratories, design better experiments, and come 
to the next svTnposium with even better scientific papers. 

The orientation of this meeting is the result of months of planning. 
There are five major topics of discussion. Each topic is being handled by 
a chairman, who is a specialist in the field, and is being introduced by a 
general survey speaker, who will cover much of the literature. The scientific 
papers are followed by discussion of the subject, which then is summarized 
by the individual chairman. The physical aspects of radiation and clinical 
studies will be discussed in a subsequent meeting. 

Our present task is a noble one, i.e., a mental cross-pollination of 
neurologists and radiobiologists interested in basic mechanisms. So without 
further comment, I w-elcome all of you and now ask you to capture your 
protons, your electrons, dendrons, axons, and neurons and orbit into new 
frontiers of learning. 

Ray S. Snider 
Symposium Chairman 



CONTENTS 

Contributors v 

Foreword ix 

Preface xi 



\ Part I. Effects of Ionizing Radiation on the 

Developing Nervous System 

Introduction to Part I 

By Samuel P. Hicks 1 

Major Radiobiolotjical Concepts and Effects of Ionizing Radiations 
on the Embryo and Fetus 

By Roberts Rugh 3 

Quantitative Histologic and Behavioral Studies on Effects of Fetal 

X-Irradiation in Developing Cerebral Cortex of White Rat 

By K. R. Brizzee, L. A. Jacobs, X. Kharetchko, and 

J. C. Sharp 27 

Structural and Behavioral Alteration in the Rat Following Cumula- 
tive Exposure of the Central Nervous System to X-Irradiation 

By Robert H. Brownson 41 

Behavioral and Histologic Effects of Head Irradiation 
in Newborn Rats 

By James N. Yamazaki, Leslie R. Bennett, and Car- 
mine D. Clemente 59 

Cytoplasmic Inclusions Containing Deoxyribonucleic Acid in the 
Neural Tube of Chick Embryos Exposed to Ionizing Radiation 

By Mary Elmore Sauer and Donald Duncan 75 

Biochemical Effects of Irradiation in the Brain of the Neonatal Rat 
By O. A. Schjeide, J. N. Yamazaki, C. D. Clemente, 

Nancy Ragan, and Sue Simons 95 

Some Effects of Nucleic Acid Antimetabolites on the Central Nerv- 
ous System of the Cat 

By Harold Koenig 109 

Geographic Distribution of Multiple Sclerosis in Relation to 
Geomagnetic Latitude and Cosmic Rays 

By John S. Barlow 123 

xiii 



xiv CONTENTS 

General Discussion 151 

Summation by Chairman 

By Samuel P. Hicks 157 

Part II. Histopathological Changes Resulting from the 
Irradiation of the Nervous System 

Basic Problems in the Histopathology of Radiation of the Central 
Nervous System 

By Orville T. Bailey 165 

Sequence of X-Radiation Damage in Mouse Cerebellum 

By NoRBERT Schummelfeder 191 

Morphological EfTect of Repeated Low Dosage and Single High 
Dosage AppHcation of X-Irradiation to the Central Nerv- 
ous System 

By Willibald Scholtz, Wolfgang Schlote, and Wolf- 
gang HiRSCHBERGER 211 

A Demyelinating or Malacic Myelopathy and Myodegeneration — 
Delayed Effect of Localized X-LTadiation in Experimental 
Rats and Monkeys 

By J. R. M. Innes and A. Carsten 233 

EflFects of High-Dose Gamma Radiation on the Brain and on 
Individual Neurons 

By F. Stephen Vogel 249 

Electron Microscope Observations on the X-Irradiated Central 
Nervous System of the Syrian Hamster 

By H. Hager, W. Hirschberger, and A. Breit 261 

Bioelectric EfTects of High Energy Irradiation on Nerve 

By C. T. Gaffey 277 

Morphologic and Pathophysiologic Signs of the Response of the 
Nervous System to Ionizing Radiation 

By N. I. Grashchenkov : 297 

General Discussion 315 

Chairman's Summation 

By Webb Haymaker 321 

Part III. Particle Irradiation of the 
Central Nervous System 

The Use of Accelerated Heavy Particles for Production of Radiole- 
sions and Stimulation in the Central Nervous System 

By Cornelius A. Tobias 325 



CONTENTS XV 

Effect of Local Irradiation of the Central Nervous System with 
High Energy Protons 

By Bengt Andersson, Borje Larsson, Lars Leksell, 
William Mair, Bror Rexed, and Patrick Sourander 345 
Production of Laminar Lesions in the Cerebral Cortex by 
Deuteron Irradiation 

By Leonard I. Malis, Jerzv E. Rose, Lawrence Kru- 

GER, and Charles P. Baker 359 

Fluorescein as a Sensitive, Semiquantitative Indicator of Injury 
Following Alpha Particle Irradiation of the Brain 

By D. C. Van Dyke, P. Janssen, and C. A. Tobias 369 

Pathologic Changes in the Brain from Exposure to Alpha Particles 
from a 60 Inch Cyclotron 

By Peter Janssen, Igor Klatzo, Jaime Miquel, Tor 
Brustad. Albert Behar, Webb Haymaker, John 

Lyman, Julian Henry, and Cornelius Tobias 383 

Some Observations of Radiation Effects on the Blood-Brain Barrier 
and Cerebral Blood Vessels 

By Carmine D. Clemente and 

Harold E. Richardson, Jr 411 

Chemical and Enzymatic Changes in Nerve Cells Irradiated with 
High Energy Deuteron Microbeams 

By Wolfgang Zeman, Howard J. Curtis, Dante G. 

Scarpelli, and Ruth Kleinfeld 429 

Tolerance of Central Nervous System Structures in Man to 
TheiTnal Neutrons 

By L. E. Farr, W. G. Calvo, Y. L. Yamamoto, E. E. 

Stickley, W. Haymaker, and S. W. Lippincott 441 

General Discussion 459 

Review of Neurophysiologic and Psychologic Research on Irradia- 
tion Injury in the U.S.S.R. 

By Walter R. Stahl 469 

Part IV. Functional Changes in the Nervous System 
Resulting from Radiation Exposure 

General Survey — Functional Changes in the Nervous System In- 
duced by Ionizing Radiations 

By Paul S. Henshaw 489 

Acute Central Nervous System Syndrome of Burros 

By Daniel G. Brown, Daniel P. Sasmore, and Larry 

R Jones 503 



xvi CONTENTS 

Effects of Low Level Radiation on Audiogenic Convulsive Seizures 
in Mice 

By Dorothea Starbuck Miller 5L3 

Effects of Ionizing Radiation on Visual Function 

By Leo E. Lipetz 533 

X-Irradiation Studies on the Mammalian Retina 

By Werner K. Noell 543 

The Effects of Ionizing Radiation on Spinal Cord Neurons 

By Makoto Sato, George Austin, and Walter Stahl 561 
Radiation Effects on Bioelectric Activity of Nerves 

By C. S. Bachofer 573 

Alteration of Mammalian Nerve Compound Action Potentials by 
Beta Irradiation 

By Edgar L. Gasteiger and Berry Campbell 597 

Action of Gamma Radiation on Electrical Brain Activity 

By Marcel Monnier and P. Krupp 607 

General Discussion 621 



Part V. Psychological Effects of Ionizing Radiation 

Effects of Radiation on the Central Nervous System and on Be- 
havior — General Survey 

By Harry F. Harlow 627 

Learning Behavior of Rats Given Low Level X-Irradiation in 
Utcro on Various Gestation Days 

By Sylvan J. Kaplan 645 

Effects of Neonatal Irradiation on Learning in Rats 

By BiLLEY Levinson 659 

Behavioral Effects of Cranial Irradiation of Rats 

By William J. Arnold 669 

Radiation-Conditioned Behavior 

By Donald J. Kimeldorf 683 

Behavioral and Correlated Hematologic Effects of Sublethal Whole 
Body Irradiation 

By T. C. RucH, Walter Isaac, and Robert W. Leary 691 
Performance of Monkeys before and after Irradiation to the Head 
with X-Rays 

By Roger T. Davis and Arnold A. McDowell 705 

Some Behavioral Effects of Ionizing Radiation on Primates 

By Arthur J. Riopelle 719 



CONTENTS xvii 

Some Effects of Radiation on Psychologic Processes in 
Rhesus Monkeys 

By W. Lynn Brown and Arnold A. McDowell 729 



General Discussion 747 

Author Index 753 

Subject Index 767 



PART I 

Effects of Ionizing Radiation on the 
Developing Nervous System 



INTRODUCTION TO PART I 



The past 10 years and especially the last few have seen a great increase 
in interest in what ionizing radiation may do to the nervous system, both 
in respect to the malforming effects induced during early development and 
the functional and structural changes occurring in later stages. Formerly, 
it was said — and sometimes still is — that the embryo brain is very radio- 
sensitive and the adult brain will stand almost anything. Unqualified, these 
phrases are almost meaningless today. We try to specify what dose of radia- 
tion produces a given effect, because one type of cell in the adult or 
embryonic nervous system may respond quite differently from another cell. 
It makes a difference whether one gives 200 r of conventional 250 kv x-rays 
to a fetal or neonatal rat in divided doses or in a single dose, or whether 
one gives a single dose of 700 r. E\en the difference in effects between 
200 r and 300 r on the fetal and neonatal cortex, cerebellum, and retina 
can sometimes be remarkable. 

The term radiosensitivity now is used more carefully, because it has 
meant quite different things to different workers. We can no longer say, 
"this stage of embryonic life is the most radiosensitive" or "that enzyme 
system is the most radiosensitive" without qualification. To a geneticist, 
radiosensitivity means that a chromosome can be changed easily; to a 
pathologist, it has often meant that a tumor cell is easily killed; and to an 
endocrinologist, it may suggest that a cells hormone production can be 
easily stopped. Many laboratories, including our own, are interested in dis- 
co\ering subtle radiation changes in neurons. For example, we are attempt- 
ing to demonstrate changes in nucleic acids in adult rat cortical neurons 
by ultraviolet microscopy following exposure to 200 or 400 r of conven- 
tional 250-kv x-rays. I note this because it reflects the growing attitude that 
we ought to be looking for obscure and perhaps totally unexpected changes 
in the nervous system and other tissues following radiation. Certainly, 
among the most attractive areas for research are those which relate to the 
roles that DNA and RNA have in both the development and, later, the 
function of neurons. 

A diverse array of approaches to problems of developmental radio- 
biology is represented in these papers on the effects of ionizing on the 
developing nervous system, including effects of successive small doses of 
radiation on neuron differentiation, chemical and histochemical changes 
in irradiated nerve cells of all ages, studies on nucleic acids of ner\e cells 



2 INTRODUCTION TO PART I 

by the use of nucleic acid antimetabolites, and considerations of disturb- 
ances of function that radiation may cause. Development is not restricted 
to embryonic life, but continues through the life of the organism, and we 
will hear not only what radiation may do long before the embryo has a 
brain, but also what man-made radiation and radiation from outer space 
may be doing during adult life. 

Samuel P. Hicks 



Major Radiobiological Concepts and Effects 

of Ionizing Radiations on the 

Embryo and Fetus 



Roberts Rugh 

Radiological Research Laboratory, 
Columbia University, New York, New York 



Introduction 

As a radiobiologist, it is appropriate to initiate this symposium witti a few 
general statements regarding the biological eflfects of ionizing radiations. We 
are concerned with ionizing radiations, not ultraviolet or infrared radiations. 
Ionizing radiations result in the excitation or loss of an electron from an 
atom, causing an unstable situation, whether it be in an atom of an inani- 
mate or animate object. Such imbalance can be brought about by alpha or 
beta particles, by x-rays or gamma rays, or secondarily by neutrons. The 
ensuing physical imbalance results in a chemical change which, in turn, may 
effect a demonstrable biologic adjustment. While the ionization may be 
immediate or instantaneous, and is in most cases undetectable by the nervous 
system, the evidence of the biologic adjustment may take decades. 

Radiations are so potent that the ionization of one molecule in 10,000,000 
is sufficient to kill almost all organisms. It matters not whether the ionization 
is brought about by any of the different qualities of radiation, the basic 
biologic reactions are the same. However, the total dose absorbed and the 
dose rate are indeed important. There is a total absorbed dose which cannot 
be sui-\'ived, and there is a dose rate so small that it can be tolerated. In all 
properly controlled radiobiologic experiments the ion density, the dose rate, 
and the total absorbed dose must be determined and recorded. In reporting 
radiobiologic experiments it should be made clear what specific organism, 
organ, tissue, or even cells are concerned, because tolerance is not uniform. 
This does not contradict the statement that the biologic reactions to ionizing 
radiations are basically the same. It does emphasize the fact that tolerance, 
restitution, and repair are properties which vary with the large variety of 
differentiated tissues. Organisms vary during their lifetime in their reaction 
to absorbed ionizing radiations, even as do cells during their process of dif- 



4 ROBERTS RUGH 

ferentiation. The radiosensitivity of a single cell may vary 1,000 times from 
its undiflferentiated to its differentiated state. The time has long since passed 
when data were adequately presented in terms of milligram hours of radium 
without consideration of the actual absorbed dose (rads), as well as the 
organ, tissue, or cell exposed. 

There is a confusion of teiTns, such as threshold, safe, permissible, or toler- 
able doses of ionizing radiations. If by threshold one means an exposure 
below which nothing happens, it is very doubtful that such a level exists. A 
single ionization occurring at a critical point on a chromosome may not 
affect its bearer, but may have permanent and drastic effects on its progeny, 
which explains the extreme caution of the geneticists. At the level of the 
atom, any effect brought about by ionizations is probably all-or-none. It is 
another matter whether such an effect is detectable and, if detectable, 
whether it is tolerable to the biologic system. Certainly some changes in the 
central nervous system of the embryo or of the adult are both detectable 
and tolerable, if by tolerable we mean that the individual is able to survive. 

At the cellular level the changes brought about by the absorption of 
ionizing radiations are irreversible, irrevocable, and irreparable, but they 
may still be tolerated by the cell. If the cell survives and is a germ cell, it 
may contribute a new mutant to its succeeding generations of cells, both 
detectable and tolerable, but not likely of any benefit. If the cell survives as 
a somatic cell, it may tolerate the damage and reproduce by mitosis for 
many years, ultimately to blossom out in two or three decades as a center of 
a malignancy. The ability to tolerate absorbed radiations is good for sur- 
vival, but possibly not good for progenies. 

The term permissible dose is used largely in Civil Defense directives and 
radiologic centers, and its dose level is generally somewhat lower than the 
tolerable. There is still another aspect of the tolerable dose, and this relates 
to the ability of the adult to repair or regenerate replacements for damaged 
tissue, usually scar tissue, or of the embi-yo to redirect its schedule and pattern 
of differentiation. The words have a wider meaning, therefore, and include 
reparative processes above the cellular level. A certain amount of radiation 
may be tolerated by an organism if the remaining tissue is sufficient for 
survival and can replace the void with protoplasmic mass. This ability is 
more evident in the embi-yo where undifferentiated cells are being directed 
and redirected after irradiation toward differentiation along the various 
routes which result in recognized tissues. Once this is completed, as in the 
adult, replacement of kind is not usual. 

A corollary of this discussion is the matter of cumulative effects. Certainly 
one can see how a primitive germ cell, successively exposed to ionizing radia- 
tions, might well accumulate mutants at various points along its chromo- 
somes. In the same way, a somatic cell may be bombarded successively by 



IONIZING RADIATIONS: EFFECTS ON EMBRYO, FETUS 5 

ionizino; radiations, and. as Ions; as it survives, it too should accumulate 
effects until an intolerable composite of the damasje would result in its death. 
In the somatic cell, exposure effects short of lethality may not be so s^raphi- 
cally demonstrable as those produced by a mutated oerm cell. Such a change 
in the somatic cell might never be detected. There is as yet no direct proof, 
but it may be conjectured that squamous cell carcinoma of the skin is more 
likely to follow repeated, accumulated, but tolerable exposures, than a single 
exposure. This presumption is based on the knowledge that squamous cell 
carcinoma, which can be produced by ionizing radiations, can result from 
tolerated exposures. 

The human embryo or fetus, and to some extent the adult, have powers of 
sloughing off the undesirable or dead cells so that the only place that cumula- 
tive effects can be detected is in the progeny of surviving somatic and germ 
cells. This is why the low and tolerable exposures are so important. To kill 
is clear cut. To maim for the duration of life may be biologically tolerable, 
but psychologically and sociologically intolerable. 

Finally, one must emphasize the difference between the somatic and the 
genetic effects. Since ionizing radiations can alter the central nervous system 
either through the germ cells or through direct irradiation, we are concerned 
with both genetic and somatic efTects. Neither is apt to be immediate; both 
can be subtle and long delayed. 

When the embryo or fetus is inadiated it must be realized that both the 
de\eloping central nervous system and the developing gonads of the organism 
may have been exposed. Congenital effects resulting from direct irradiation 
of the de\eloping organ primordia cannot have genetic corollaries except by 
coincidence, which is \ery unlikely. Congenital effects following direct 
irradiation cannot be inherited. However, concomitant with somatic exposure 
there may be germ cell exposure which may well result in different and 
possibly e\en more severe effects, but ones which cannot become apparent 
until they appear in a succeeding generation. Somatic exposures alone may 
alter the soma of one generation, but germ cell exposures may alter all suc- 
ceeding progenies. 

Effects on the Embryo or Fetus 

In analyzing the embryonic effects one must have in mind lour special 
situations: 

1. The medium of the embryo is acjuatic. and there is reason to believe 
that this enhances its radiosensiti\ity. 

2. The embryo is a mosaic of acti\ely differentiating centers with con- 
stantly changing but high mitotic indexes, both conditions enhancing radio- 
sensitivity. 



ROBERTS RUGH 



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IONIZING RADIATIONS: EFFECTS ON EMBRYO, FETUS 

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8 ROBERTS RUGH 

3. The embryo can be killed by irradiations which for any species are less 
than the lethal dose for the adult, and consjenital effects may be produced by 
exposures of about 2^,r that of the LD/50/30 level (Figs. 1-27). 

4. If the embryo survives the irradiation, it has powers of topographic 
repair which are not known to the adult. But it cannot step up cell produc- 
tion to replace cells lost trom radiation necrosis, so that the net result is a 
deficient embryo or fetus — deficient in those cells or those tissues which are 
most damaged at the time of irradiation (Figs. 28-35). 

Every one of its systems may be affected by exposure of the embryo or 
fetus to ionizing radiations, but the most obvious effects appear to be on the 
central nervous and the skeletal systems. Probably the most common and 
graphic effect, one most frequently reported from Hiroshima and Nagasaki, 
as well as in experimental radiobiology, is microcephaly. This is not an 
isolated condition, and all those so affected undoubtedly exhibit other 
anomalies. There is often stunting, microphthalmia, and loss or reduction of 
other parts indicating deficits (Figs. 21-35). 

But anomalies designated as congenital may be caused by irradiation at 
times other than during differentiation. First, exposure of the sperm cell or 
its precursor may produce the anomaly in all succeeding generations due to 
chromosomal effects. Second, irradiation of the ovary may cause the anomaly 
to appear in successive generations. Third, the embryo is most likely to de- 
velop specific anomalies if the differentiating organ concerned is irradiated 
directly. Fourth, the embryo at any time from the moment of fertilization of 
the egg through the completion of organogenesis may be caused to develop 
the same type of anomaly. Once organogenesis is completed, congenital 
anomalies can no longer be caused by irradiation (Figs. 1-12 and 17-20). 

Thus, congenital anomalies involving the central nervous system may be 
caused by irradiation of either germ cell, of the actively differentiating 
organism, or of any stage prior to this. 

In our studies we have concentrated on the cerebral hernia or exen- 
cephalous condition where the midbrain protrudes through the cranial roof. 
This is a graphic and readily observable maldevelopment, and any fetus 
exhibiting this condition is presumed also to have other, possibly less graphic, 
but even more serious effects. Since this anomaly is readily observable, it has 
been a convenient marker of severe irradiation damage to the developing 
embryo (Fig. 21 and Table I). 

Exencephalia has been produced by the irradiation of the mouse testis or 
ovaiy and has appeared in successive generations following a single exposure. 
True, its frequency is very low, but it is a severe and lethal anomaly which 
can be genetically produced. It has also been produced by exposing the 
mouse embryo at any time from fertilization through gestation day 8.5, and 
in the earlier stages with doses of as little as 15 r. Exposures of 5 r at certain 



IONIZING RADIATIONS: EFFECTS ON EMBRYO, FETUS 9 

periods have increased the intrauterine mortality by lO'^r, so that in the 
early embryo we are probably dealing with the most radiosensitive stage in 
ontogeny. No exencephalies ha\e appeared among thousands of unirradiated 
control embryos or in those irradiated after completion of organogenesis. It 
has been produced, however, by other traumatic conditions (Figs. 19, 20. 
22.24). 

The term low dose should be defined here. Green ( 1959), the geneticist, 
says: "There is no totally sate dose of radiation," so that to him there is no 

TABLE I 

Malformations .Among Normal Offspri.ng of CFi X CFi Mice ' 



Offspring Xumber Per cent 



Pregnancies 61 



Total embryos 630 

Normal embryos 591 94 

Dead embrvos 2 0.3 

Resorptions 37 5.7 



Exenccphal 



les 



* This table shows that among 63(1 unirradiated CFi mouse embiyo>, 
not a single exencephaly (brain hernial developed. However, note that 
there were almost 6% resorptions and two dead embryos. This is an 
expected ratio and may be due to genetic causes. \t no time in our 
experience, while examining thousands of mouse embryos. ha\e we 
found the congenital anomaly of e.xencephaly among the control mice. 



Figs. 1-12 are on pages 6 and 7. 

Figs. 1 and 2. These are normal mouse eggs seen during the first 24 hours after 
conception. Fig. 1 shows the egg at the moment of sperm entrance and Fig. 2 shows 
the two pro-nuclei and the first polar body. Highest percentage of resorption follows 
irradiation at this stage. 

Fig. 3. This shows a group of normal mouse embryos at 1.5 days in the 2-cell 
stage. This is probably the most radiosensiti\e period with respect to the production 
of irradiation congenital anomalies. 

Fig. 4. This is a mouse egg at 1.5 days which had recei\ed 50 r at 0.5 days and 
shows a hyperchromatic nucleus. 

Figs. 5 to 8. These show various stages in the disintegration of the mouse embryos 
following exposure to 15 r at 1.5 days. Note that in some cases the pro-nuclei are 
being extruded from the cytoplasmic mass. It is unlikely that the residual cellular 
material could survive. Howc\er. the majority of eggs exposed at this time and to 
this level of irradiation would survive. 

Figs. 9 to 12. These are all irradiated embryos exposed to 15 r at 1.5 days and 
examined at 2.5 days. Note pyknosis, hyperchromatism, and fragmentation of the 
embryos. 



10 



ROBERTS RUGH 




IONIZING RADIATIONS: EFFECTS ON EMBRYO, FETUS 



11 



HP « 




12 ROBERTS RUGH 

such thing as a low dose, every exposure is too high. In our times this may 
be impractical and unrealistic, though still a concept to be respected. 

In Copenhagen, Dr. Hammer- Jacobson (1959) states: "Fetal doses of 
less than about 1 r are presumed to cause no noticeable injury. . . . Fetal 
doses between 1 r and about 10 r are assumed in some instances to cause 
injuries in the form of diseases, malformations, slow development, or reduced 
resistance, especially when the irradiation occurs between the 2nd and the 
6th week. ... If there are additional indications, therapeutic abortion 
should be assumed advisable. . . . Fetal doses above about 10 r are assumed 
to involve a rather great probability of fetal injury. In such cases induction 
of abortion should therefore be the general rule." (See Table II.) 

In all of this, reference is made to the somatic efTects, but certainly the 
geneticist would concur. A whole body exposure of as little as 12 r will cause 
some lymphopenia in the adult; and Brues (1959) refers to 25 r as a "low 
dose" for somatic effects, probably because there are always at least some 
hematologic changes. Thus, a dose of 5 r may not have measurable conse- 
quences for the somatic tissues of the adult, but would be seriously damaging 
to the embryo or to its progeny through effects on the gametes. 

The embryo or fetus is not simply a miniature of the adult and must be 
regarded as a dynamic, tirelessly changing mosaic of differentiating areas all 
integrated into an over-all pattern under organismic influences which appear 
themselves to be immune to ionizing radiations. As long as there are undam- 
aged building units for development which are adequate in number and 
basically intact, these influences will attempt to organize them into a topo- 
graphically normal, balanced embryo. But the undamaged cells cannot 
replace those that were killed by irradiation. Any stimulus to excess cell pro- 
duction is cancerogenic, so that the embryo may be topographically well 

Figs. 13-20 are on pages 10 and 11. 

Fig. 13. This shows 3 normal mouse blastulae suspended within the uterus at 3.5 
days. 

Fig. 14. This shows the normal mouse embryos at the moment of implantation at 
4.5 days. 

Figs. 15 and 16. Mouse embryos at 4.5 days (time of implantation), but following 
x-irradiation with 15 r at 1.5 days. They show pyknotic nuclei, discarded (necrotic) 
cells within the blastocoel, and failure at implantation. These embryos might survive 
to give rise to deficient fetuses. 

Fig. 17. This embryo was likewise exposed to 15 r at 1.5 days and exhibits a giant 
ceil with prominent chromosomes at 4.5 days. This is a common irradiation sequela. 

Fig. 18. This is a mouse embryo treated as that of Fig. 17, showing a prominent 
cell with vacuolization. This is a frequent irradiation consequence. 

Figs. 19 and 20. These are members of litters dissected at 18.5 days showing 
stunting, anencephaiy, and exencephaly, while other members of the litter appear 
superficially to be normal. When compared with controls they, too, are shown to be 
stunted. 



IONIZING RADIATIONS: EFFECTS ON EMBRYO, FETUS 

TABLE II 

Anomalies Reported Following Himan Fetal X-Irradiation ^ 



13 



1. Microcephaly (most frequent) 

2. Hydrocephalus 

3. Poroncephaly 

4. Mental deficiency 

5. Mongolian 

6. Idiocy 

7. Head ossification defects 

8. Skull malformations 

9. Micromelia 

10. Microphthalmus 

11. Microcornea 

12. Coloboma 

13. Strabismus 

14. Cataract 

15. Chorioretinitis 



16. 


Nystagmus 


17. 


Stillbirth increase 


18. 


Decrease live birth weight 


19. 


Neonatal and infant death increase 


20. 


Ear abnormalities 


21. 


Spina bifida 


22. 


Cleft palate 


23. 


Deformed amis 


24. 


Clubfeet 


25. 


Hypophalagism 


26. 


Syndactyly 


27. 


Hypermetropia 


28. 


Amelogenesis 


29. 


Odontogenesis imperfecta 


30. 


Gf-nital deformalities 



■'' This table lists thirty coiigcnital anomalies found in humans following fetal .\-ii radiation. .Vote that 
the most frequent type of anomaly relates to the central nervous system. Most, if not all of these 
anomalies, have been produced in experimental animals by exposure during embryonic development. 

'' It must be remembered that the levels of irradiation which are hazardous for the embryo or fetus 
aie very much lower than those foi the somatic tissues of the adult organism. It is, therefore, obvious 
that extreme caution should be exerted where either the reproductive organs or the developing embryo 
might be involved. We do not yet know the extent or the duration of radiation effects on the fetus or 
the germ cells. 



TABLE III 

Effect of Low-Dose X-ravs on the Early 
MorsE Embryo ' '' 



^ 





S 
o 


o 






9.6 


94.0 


5.7 


0.3 


0.0 


10.0 


85.0 


15.0 


0.0 


0.0 



Controls 

5 r at 1 .5 dav 



630 

80 



» This table presents data following 5 r exposure of the mou.se embryo at !'/> days post conception. At 
this time, the mouse embryo is in the 2 cell stage. Eighty such embryos exposed to 5 r gave 15% 
resorptions which was almost a W7( increase over the expected 5.70^ of the controls. No exencephaly 
appeared. 

*> .\n inciease of ').W, in intrauterine deaths caused by 5 r exposure at 1-2 cell stage. 



14 



ROBERTS RUGH 




IONIZING RADIATIONS: EFFECTS ON EMBRYO, FETUS 15 




16 ROBERTS RUGH 

TABLE IV 
X-Irradiation of the Early Mouse Embryo * 



^ 



c< 



iii 



i^ 



0.5 52 58 42 

1.5 90 95 5 

2.5 95 73 24 3 

3.5 76 88 9 3 

4.5 53 92 8 

5.5 37 77 17 6 

6.5 77 92 8 

7.5 25 96 4 

8.5 51 96 4 

9.5 12 90 10 



568 85.7 12.3 2.0 

" This table gives data from an extensive study of the effect of 50 r x-iays on the mouse embryo at 
various days from 0.5 to 9.5. In any somatic study 50 r would be considered a low level exposure, but 
from this study, when such an exposure kills 42% of the embryos at 0.5 days and large percentages at 
2.5, 5.5, and 9.5 days, it is obvious that 50 r to the early developing mouse embryo is a high level 
of exposure. Of course, exencephalia was produced, the largest per cent being at 5.5 days. 



Figs. 21-27 are on pages 14 and 15. 

Fig. 21. An enlarged \'iew of exencephaly (brain hernia) in the mouse. This is a 
protrusion of the mesencephalon through the cranial roof. 

Fig. 22. Three members constituting an entire littt r, all .showing severe exencephalic 
maldevelopment. This followed exposure of 50 r at 2.5 days. 

Fig. 23. Note the same group of 3 congenital anomalies in a field including a 
normal control mouse fetus of the same age. This demonstrates that in addition to 
congenital anomalies there is often a stunting of the irradiated embryos. 

Fig. 24. This shows an entire litter, as found in the bicornate uterus of the mouse 
at 18.5 days, following an exposure of 200 r at 8,5 days. Note that 5 of the 11 litter 
members exhibit exencephalia. 

Fig. 25. This shows 4 members of a litter exposed to 50 r at 3.5 days. These are 
to be compared with a single control above. Note not only congenital anomalies but 
stunting of every member. 

Figs. 26 and 27. These mice were exposed to 50 r fractionated to 25 r each at 
two times during embryonic development, one exposure occurring before implantation 
and the second after implantation but before the completion of neurogenesis. Note the 
bizarre form of the extruded mesencephalon. The 2 litter members appear to be normal 
but are stunted. 



IONIZING RADIATIONS: EFFECTS ON EMBRYO, FETUS 17 

balanced, but at the same time may exhibit sross deficiencies. The brain 
may appear to be grossly normal, but when compared with the control brain 
may be seen to be microcephalous. Once the neuron is differentiated, it is 
then almost completely radioresistant, but neurosenesis is not completed by 
the time of birth. Thus, irradiation effects on the central ner\ous system 
extend from the serm cell throus^h the completion of neurogenesis of the 
next generation, at least. Put more succinctly, ionizing radiation should be 
respected by germ cells at all times and by all undifferentiated cells i Tables 
III and IV). 

In treatment of central nervous system malignancies, doses ot 10.000 r are 
sometimes accumulated. If killing a tumor by irradiation results in the saving 
of a life, it is certainly justified. If it results in the prolonging of a life with 
concomitant and permanent injury and possible germ cell exposiuc. the 
procedure may be questioned. When the indi\idual is beyond the reproduc- 
tive age, there is no place for this discussion. The emphasis here is on the 
germ cells which might be used, and on the embryo or fetus which should 
never be exposed if it can be avoided. Any exposure of the germ cells or early 
embno is undesirable. 

Gentry ct al. (1959i ha\"e found a correlation between the areas in New 
York State of high congenital malformations and geographic concentrations 
of natural materials of relatively high levels of radioactivity, such as igneous 
or black shale rock. These may include C'*, K^", Ra--''. Th" ■-', and U- •\ and 
their decay products. The a\erage exposure of indi\iduals was estimated at 
2.1 to 3.2 r per 30 years. The highest record for any single town was 66.7 
congenital malformations per 1,000 births, and in an area particularly high 
in natural radioacti\ity. This was considerably above the average in the 
"unlikely areas" of 12.9 per 1,000 live births. There were some towns with 
no anomalies in low radiation areas. A reduction in birth weights also showed 
a correlation with increasing radioacti\ity. Detailed maps of radioactive 
concentrations fitted perfectly those of higher incidence of congenital mal- 
formations. Of all malformations. 15''r involved the central ner\ous svstem. 
and of these 94.5''r caused death. While some may doubt the conclusions of 
this study, it cannot be ignored. 

A somewhat similar study has been made by Wesley i 1960) in which he. 
as a statistician, finds that "96'' r of all deaths due to congenital malforma- 
tions are caused by background radiation, and x-rays have caused a 6''r 
increase in congenital malformations in the United States in the last 30 
years."" There was a low incidence of congenital anomalies in southeastern 
.Asia and a high one in northern Ireland, correlated with background con- 
centrations. There is no way of determining how manv of the 5.000.000 
mentally retarded United States citizens are products of irradiation injury. 

Hicks ct al. i 1959) made the following statements, all of which emphasize 



18 



ROBERTS RUGH 




IONIZING RADIATIONS: EFFECTS ON EMBRYO, FETUS 19 




20 ROBERTS RUGH 

that fetal irradiation of the rat results in a neurologically deficient embryo: 
"The cerebral hemispheres and diencephalon were a a^ood deal smaller 
than normal. . . . The neocortex was seriously deficient, and about half as 
thick as normal at the vertex to about 2^ normal thickness laterally. . . . 
Small pallium. . . . The anterior commissure was a little less compact. . . . 
The midbrain was smaller in total cross area than normal due to somewhat 
flattened superior colliculi. . . . The cerebellum was altosjether a little 
smaller than normal. . . . The lower medulla showed a slight reduction 
in total o\er-all size. . . . The lower brain stem and cerebellum were a little 
smaller than normal. . . . The cords were a little smaller in cross section 
than normal. . . . Most of the cells in the 13-day retina were killed by 
radiation." 

Anomalies mentioned in this excellent study included: 

"Radiation-killed cells in the periependymal primitive matrix threw the 
mitotic layer into rosettes which continued to proliferate brain, nonetheless. 
The result was an anomalous mass of ectopic cortex. . . . No corpus cal- 
losum. . . . Bizarre bundles of fibers. . . . Disorderly array of all sorts of 
cortical neurons. . . . The neurons were jumbled, scattered, and they were 
often upside down or pointed sideways." 

Figs. 28-35 are on pages 18 and 19. 

Figs. 28, 29, and 30. These represent members of litters from 3 successive genera- 
tions following a single exposure of the ovary of the mother of those in Fig. 28 to 
100 r. It was whole-body e.xposure, but we have reason to believe that the somatic 
eflFects of this exposure had nothing to do with these congenital anomalies. The fact 
that a single exposure of the ovary caused this brain anomaly to appear in three suc- 
cessive generations is genetically significant, even though its incidence was very low. 

Fig. 31. These represent an entire litter from a grandfather who had received high- 
level exposure of his testis. The first generation appeared normal, were viable and 
fertile. This brain anomaly of exencephalia appeared in the next generation. One 
might expect it to appear in yet succeeding generations. 

Fig. 32. The four embryos to the right show the variety of anomalies which ap- 
peared in the second generation following testis exposure. All were stunted, some died 
as fetuses (late in development). The single member to the left is a control of the 
same age. 

Fig. 33. When mouse embryos are exposed after organogenesis to high but tolerable 
levels of irradiation, the eflFects are largely skeletal. Note particularly the variations in 
size within the single litter. One member of the litter is almost as large as the controls. 
The explanation of this is probably genetic. 

Fig. 34. This is one litter, all of whom were exposed at the same time to the same 
irradiation, but which exhibit a wide range of difference in size. 

Fig. 35. This shows photographs of Spalteholz's preparations of two mice at birth, 
the upper one being the control, the lower one x-irradiated at 13.5 days. Note that 
the irradiated embryo appears to be topograhically normal but obviously is very much 
stunted. The developmental processes have been able to reorganize the undamaged 
cells to provide an apparently normally proportioned but stunted mouse. 



IONIZING RADIATIONS: EFFECTS ON EMBRYO, FETUS 21 

All of these anomalies could be attributed to deficiencies during develop- 
ment caused by ionizing radiations. 

Their explanations of these central nervous system malformations included 
the following direct statements: 

"There was a selective extirpation effect on certain primiti\e cells. . . . 
A patchy deficiency of cells. . . . Numerous dead cells spilled into the 
ventricles. . . . Virtually all of the primitive migratory cells in transit were 
killed. . . . The residual mitotic colony of lining cells was thrown into dis- 
order because their support, the matrix of radiosensiti\e cells forming much 
of the wall, was gone." 

The entire emphasis of this study seemed to be on the deficiencies follow- 
ing fetal irradiation. 

The appearance of rosettes has often been described in both the neural 
retina and in the de\eloping cortex following fetal irradiation. The presence 
of rosettes is proof that cells have been desegregated and that those still 
viable attempt neural organization. In the case of irradiation, the desegrega- 
tion is due to the killing of radiosensitive cells, which are then removed, 
leaving loosely scattered, but \iable cells. It has been shown recently (Mos- 
cona, 1960) that presumptive nerve, cartilage, and liver cells of mouse and 
chick embryos may be desegregated (disaggregated) by trypzinization and 
mixed together, only to reaggregate with respect to whether they were 
nervous, cartilage, or liver, and irrespective of whether they came from the 
mouse or the chick. In other words, presumptive ner\e cells show an aflfinity 
for eacli other, regardless of their genetic source. When they come together 
without sustentacular materials, they tend to form rosettes which are an 
expression of disorganization. The rosettes are therefore not a peculiarity of 
post irradiation, nor of the mouse or rat, but rather of neural disorganization. 
A single rosette has been formed of neuroblasts from both the mouse and 
the chick embiyos. These structures, usually temporary in the irradiated and 
developing embryo, simply represent a stage in the reorganization of viable 
nerve cells which are inadequate in number to accomplish structural 
normality (Figs. 36-43 and Table V). 

Our current studies are utilizing low doses to determine the effect of 
ionizing radiations on the developing central nervous system as demonstrated 
by beha\ ior, electroencephalographic records at \ arious stages of maturation, 
and electron microscope and neuropathologic studies of the postnatal brain. 
It may develop that it will be the experimental psychologist who will spot 
the specific developmental stages most drastically affected by ionizing radia- 
tions. If our society is primarily concerned with the function of the central 
nervous system, we may be dealing with radiation changes which are beyond 
analysis by the conventional neuropathologic techniques or by the electron 
microscope. We expect to have information on this during the next year. 



22 



ROBERTS RUGH 













IONIZING RADIATIONS: EFFECTS ON EMBRYO, FETUS 



23 



♦ • / 




SVvC*r>^/2^, •- 



'9^-^- 




•p*ystii.^-^ 




24 ROBERTS RUGH 

TABLE V 

Effect of Fetal X-Irradiation on Mouse Eyes ' 
(Measurements at 6 weeks of age) 

Average diameter (in mm) '' Relative volume (%) 

Controls 3.555 100 

150 r at 12.5 days gestation 2.970 69.6 

250 r at 12.5 days gestation 2.670 50.6 

" X-irradiatioii f>f tlic developing mouse embryo seems to result in cellular deficiencies because tlic 
damaged cells aie removed. When these ii radiations occur early, before the development of a specific 
organ system, the deficiency resulting from the elimination of the necrotized cells results in a reduction 
of organ size. The data of Table IV show that uith increasing irradiation at 12.5 days, there is a 
decrease in the relative volume of the diameters of the mouse eyes at 6 weeks of age. An exposure of 
250 r reduced the volume to approximately 50'J^. There are no studies thus far relative to the visual 
acuity of these eyes. 

'' Minimum of 8 diameters of fixed eyes taken foi each a\eiage. 

Summary 

The early embryo is more radiosensitive than is the organism at any other 
time in its entire life cycle. The earlier the stage, the more sensitive, with re- 
gard to both survival and the development of anomalies. 

At the cellular level, there is no such thing as "recovery" from irradiation 
damage, meaning a return to the preirradiated state. Since embryonic cells 
are precursors of all cells of the adult, irreparable damage to surviving cells 
results in such damage to all descendant cells of the adult organism. Ionizing 
radiations represent a very potent tool. 

Figs. 36-43 are on pages 22 and 23. 

Fig. 36. When mouse embryos at 6.5 days are exposed to x-rays, 24 hours thereafter 
they show the sloughing off of cells into the central cavity as seen here. The inner 
neurectoderm will be deficient to the extent of this cellular loss. 

Fig. 37. This shows the neural groove at the level of the brain of 8.5-day embryos 
24 hours after exposure to x-rays. Note the many pyknotic nuclei and the sloughed off 
cells into the neural groove. 

Fig. 38. This is similar to Fig. 37 except it is at the level of somites. 

Fig. 39. In this figure note the many phagocytes posterior to the developing retina, 
each of which contains a number of necrotic neurectoderm cells. This occurs about 
24 hours after x-irradiation, but the retina will be deficient to the extent of this 
cellular loss. 

Fig. 40. This is an enlarged view of the retina 4 hours after irradiation, showing 
many pykotic nuclei. 

Fig. 41. This is an enlarged view of single phagocyte containing 14 dead neurecto- 
derm cells from the x-irradiated retina. 

Figs. 42 and 43. These are enlarged views of the retina of the control Fig. 42 and 
the irradiated Fig. 43 to show slight thinning of the various layers in the x-irradiated 
eye of the mouse. 



IONIZING RADIATIONS: EFFECTS ON EMBRYO, FETUS 25 

The embryo, in contrast with the aduh, has powers of reoroaniziny; its re- 
sidual and surviving cells so that topographic normality may be achieved. 
However, every such indi\idual will be deficient, either in parts or in the 
stunting of the whole. 

Deficiencies are seldom similar in litter mates, owing to the submicro- 
scopic nature of ionizing radiations, the genetic \ariations in individuals, 
the \arying abilities for restitution, and probably other factors. 

Irradiation of the embryo is the only way to produce irradiation congenital 
anomalies, but such anomalies may be produced by other traumatic means. 
Following organogenesis, irradiation efTects are similar to those one expects 
in the adult. 

The embryo after a certain stage possesses gonad primordia or developing 
gonads, and these are subject to irradiation effects which may not be evident 
for generations. 

Central nervous system anomalies may be produced by irradiation of the 
mature gamete of either sex, the fertilized egg, or any stage in development 
prior to completion ot neurogenesis. Some formati\e cells are present even 
after the birth of the mammal. The range of radiosensitivity of gamete to 
formed organism is such that discussion of threshold is meaningless. We can- 
not now state the extent or the duration of irradiation damage to the de- 
veloping central nervous system. There may well be subtle effects to be 
revealed by population studies o\er generations. Any exposure of the early 
embryo should be regarded as too much. 

Finally. I would like to make four specific requests: 

1 . l^hat we insist on better and more adequate controls in radiobiology. 

2. That radiation dosimetry in all radiobiologic experiments be checked 
by a qualified radiophysicist and be fully reported. 

3. That there be a pool of research information on neurologic effects, in- 
cluding critically reviewed information fiom the U.S.S.R. because of the 
language barrier. 

4. That symposia of this sort be organized as frequently as the accelerat- 
ing accumulation of data demands. 



References 

Brues, .\. M. (ed.) 1959. Low-k-\el irradiation. Publ. Am. A'isoc. Advance. Sci. 59. 

Gentry. J. T.. Parkhurst, E., and Bulin, G. V. 1959. .\n epidemiological study of con- 
genital malformations in New York State. Am. J. Public Health 49, 1-22. 

Green, E. L. 1959. Genetic efTects in low-lc\fl irradiation. Pub!. Am. A^wc. Advance. 
Sci. 59. 

Hammer-Jacobson. E. 1959. Therapeutic abortion on account of x-ray examination 
during pregnancy. Danish Med. Bull. 6, 113-121. 



26 ROBERTS RUGH 

Hicks, S. P., DAmato. C. J., and Lowe. M. J. 1959. The development of the mam- 
malian nervous system. I. Malformations of the brain, especially the cerebral cortex, 
induced in rats by radiation. II. Some mechanisms of the malformations of the 
cortex. /. Comp. Neurol. 113, 435-469. 

Moscona, A. 1960. Private communication, in press. 

Rugh. R. 1959a. Vertebrate radiobiology (embryology). Ann. Rev. Nuclear Sci. 9. 
493-522. 

Rugh, R. 1959. Ionizing radiations: Their possible relation to the etiology of some 
congenital anomalies and human disorders. Military Med. 124, 401-416. 

Rugh, R., and Grupp, E. 1959a. X-irradiation exencephaly. An^. J. Roentgenol., Ra- 
diurn Therapy Nuclear Med. 81, 1026-1052. 

Rugh, R., and Grupp, E. 1959b. Exencephalia following x-irradiation of the pre- 
implantation mammalian embryo. /. Neuropathol. Exptl. Neurol. 18, 468-481. 

Rugh, R., and Grupp, E. 1959c. Response of the very early mouse embryo to low 
levels of ionizing radiations. /. Exptl. Zool. 141, 571-587. 

Rugh, R., and Grupp, E. 1960. Protection of the embryo against the congenital and 
lethal effects of x-irradiation. Atompraxis 6. 209-217. 

Runner, M. N. 1959. Metabolic Mechanisms of Teratogenic Agents During Morpho- 
genesis, Natl. Cancer Inst. Monograph No. 2 (Symposium on Normal and Ab- 
normal DiflFerentiation and Development). 

Wesley, J. P. 1960. Background radiation as the cause of fatal congenital malforma- 
tions. Intern. ]. Radiation Biol. 2, 97-1 18. 



Quantitative Histologic and Behavioral 

Studies on Effects of Fetal X-lrradiation in 

Developing Cerebral Cortex of White Rat * 

K. R. Brizzee. L. a. Jacobs. X. Kharetchko. and J. C". Sharp 

University of Utah College of Medicine, 
Salt Lake City, Utah 

Introduction 

Recent experimental studies on effects of fetal irradiation on nervous tis- 
sues have clearly showns (Hicks, 1954. and Hicks it al., 1957 i that primiti\e 
neuroblasts and spongioblasts are selecti\ely damaged by ionizing i adiation. It 
has also been demonstrated in this work that rather specific and predictable 
anomalies are produced in nervous tissues in postnatal life in the rat by radia- 
tion exposure on any given day in the gestation period between the 9th day and 
birth. At the same time, some beha\ioral studies i Levinson. 1952: Furchtgott 
and Echols. 1958a. bi have demonstrated serious beha\ioral deficits in rats 
irradiated as fetuses. While considerable attention has been given in the 
histopathologic studies to the regenerati\e ability and reco\ery of nervous 
tissues from such radiation exposure (Hicks. 1957) little effort has been 
devoted to analyzing the capacity of the cells sur\iving irradiation exposure 
for normal growth or the specific effects of the irradiation on cell growth. 
Further, the behavioral studies carried out thus far have not emphasized 
the possible relationships between cytologic deficits and beha\ ioral deficits. It 
has been our purpose, therefore, in initiating the present series of investiga- 
tions to analyze effects of fetal x-irradiation administered in fractionated 
and single doses on early postnatal growth of sur\i\ing cells in cerebral 
cortex and to determine what relationships may exist between alterations in 
normal growth patterns or cytologic deficits and beha\ioral abnormalities 
appearing later in life. The present report is concerned with our preliminan- 
findings with fractionated doses administered during the latter half of the 
testation jx-riod. 

* Supported in part by research grants from the National Institute of Neurological 
Diseases and Blindness. National Institutes of Health, and the University of Utah 
Research Fund. 

27 



28 BRIZZEE, JACOBS, KHARETCHKO AND SHARP 

Materials and Methods 

Three groups of rats of the Sprague-Dawley strain were exposed in utero 
to fractionated doses of total body x-irradiation. 

Two of the groups received 12.5 and 25 r per day at the rate of 60 r per 
minute on gestation days 10 through 17 giving total doses of 100 and 
200 r, respectively. A third group (Brizzee et al., 1961) received a total 
dose of 300 r given at 60 r per day on gestation days 10 through 14. These 
animals and a series of control animals treated in the same manner as the 
above groups, except for the exjx)sure to radiation, were grouped according 
to age at 1,5, 10, and 20 days with from 4 to 6 animals per group and the 
sexes equally divided. The tissues were fixed and stained as reported previ- 
ously (Brizzee and Jacobs, 1959; Brizzee et al., 1961) and subjected to 
quantitative histologic analysis. The parameters studied were neuron packing 
density, neuron nuclear, cytoplasmic, and soma volume, nucleocytoplasmic 
ratio, gray cell coefficient, glial packing density, and the glia/neuron index 
in area 2 (Krieg, 1946). In addition, total brain weight was determined and 
cortical thickness measured in areas 2, 4, 41, and 17 (Krieg, 1946). All of 
the volumetric, density, and thickness determinations were confined to the 
submolecular layers only. Methods employed in the quantitative histologic 
determinations have been described in earlier publications (Brizzee and 
Jacobs, 1959; Brizzee r^ ai, 1961). 

In the behavioral studies, 9 pregnant rats were divided into three equal 
groups: a full-body group, a half-body group in which the lower half of the 
dam was shielded by lead, and a control group which received no irradiation. 
Irradiation took place each day from the 10th through the 17th days of 
gestation. Each irradiated animal received 40 r per day for a total of 320 r 
(60 r per min). For the half-body group, a shield made of blocks of lead 
2 X 4X 8 in. was constructed so that only the thorax, neck, and head were 
exposed to radiation. 

To assess the duration of the effects of x-irradiation on locomotor coordi- 
nation, the three groups of rats were divided into three subgroups to be 
tested at different ages. Group one was tested at age 40 days, group two at 
age 90 days, and group three at age 140 days. The test of locomotor coordi- 
nation required the rats to traverse a bridge made of two parallel rods. 

At 115 days of age the rats in the 90- to 140-day groups were given 2 
trials in a simple L-shaped water maze. The next day all the rats were run 
in a 14-unit multiple-T water maze patterned after the Stone design (Heron, 
1930; Sharp, in press). 

At age 50 days 6 rats from each group were sacrificed, and their cerebral 
cortexes studied in the same manner as in the first three groups. 

In plotting the values of the various parameters in Figs. 1-6 the vertical 



FETAL X-RAY EFFECTS: CEREBRAL CORTEX 



29 



lines indicate the maa;nitude of the standard errors of the mean for the con- 
trols. Standard errors for the irradiated s^roups were not plotted in the 
interest of clarity in the figures. 

Results 

Neuron packing density in all groups i Fig. 1 ) was seen to decrease very 
rapidly between the 1- and 5-day stages, with a less notable decrease between 
the 5th and 10th days, and approached normal adult levels on the 20th day. 
The values for all irradiated groups and controls were in close agreement 
and showed no significant differences at anv asje level. 



10.0 



9.0 



ro 


RD 


t- 




E 




If) 




O 


7.0 


X 




• — 




V 




1- 


6.0 


00 




2 




III 




o 




o 


5.0 


z 




:^ 




(J 




S 


4.0 


?- 




o 






3.0 


UJ 





2.0 



1.0 



I- CONTROL 
^ - lOOr 
= 200r 
o = 300r 




5 10 

AGE (DAYS) 
Fig. 1. Early postnatal changes in neuron packing density. 



20 



30 



BRIZZEE, JACOBS, KHARETCHKO AND SHARP 



The mean values for neuroglial packing density in the control groups 
decreased from 45,000 cells/mm^ at 1 day to 27,000 at 20 days, but the dif- 
ferences between the various age levels in this series are not significant owing 
to a rather large variance in counts. The mean neuroglial packing density 
for all four age groups in the nonirradiated animals was 34,000 cells/mm^. 
The neuroglial density in the 100 and 200 r groups did not differ significantly 
from the controls, but in the 300 r group, the value for neuroglial density in 
1 -day-old animals was significantly higher (80,000 cells/mm'; /; < .05) 
than in the nonirradiated groups. In later stages the differences were not 
significant. 

The neuroglia/neuron index (Fig. 2), almost entirely as a result of the 



0.40 

0.38 

0.36 
034 

0.32 

030 

0.26 

xO-26 

UJ 

00.24 

z 

022 

z 

S020 

D 
UJ 

z 0.18 
< 0.16 
§0.14 

010 
0.08 
0.06 

0.04 
0.02 




1= CONTTROL 
A^ lOOr 
o-200r 
o- 300r 



10 
AGE (DAYS) 



20 



Fig. 2. Increase in neuroglia/neuron index from 1st to 20th postnatal day. 



FETAL X-RAY EFFECTS: CEREBRAL CORTEX 



31 



changes in the neuronal packing density, increased fairly rapidly in controls 
from the 1st to the 10th day and more slowly from the 10th to the 20th day. 
Differences in values for the neuroglia/ neuron index between irradiated 
and control groups were not significant at the 5-, 10-, and 20-day stages. In 
the 300 r group, however, the neuroglia/neuron index in the 1 -day-old rats 
is significantly higher ip < .05) than that in the non-irradiated rats of the 
same age. In 20-day animals the value for the index in the 200 r group is 
considerably higher than in the controls, but due to a large variance it is 
not statistically significant at the 0.05 le\el. 

It is noteworthy that the levels for the neuroglia/neuron index at all ages 
studied are very low as compared with adult values in some other species as, 
for example, in man (1.78; Hawkins and Olszewski, 1957) or in the horse 
(1.24; Friede, 1954), although the index is comparable in our 20-day 
animals to the average values derived from Friede's data for the cerebral 
cortex in the mouse (.35) and the rabbit (.42). 

Neuron nuclear, cytoplasmic, and soma (nucleus -\- perikaryon) volumes 
(Fig. 3) increased steadily from the earliest to the latest stage examined with 
the values in all groups in fairly close agreement. As in our preliminary 
studies describing the 300 r group ( Brizzee ct oL, 1961), however, the 



1400 



1200 



:jlOOO 



^ 800 

o 
> 



600 



6 400 
cr 

3 



200 




I - CONTROL 
A = lOOr 
° = 200r 
300r 



10 
AGE (DAYS) 



20 



Fig. 3. .\lterations in neuron soma volume in early postnatal stages. 



32 



BRIZZEE, JACOBS, KHARETCHKO AND SHARP 



2.0 


( 


\ 


L8 




,\\\ I = CONTROL 




• 


\aA a = lOOr 
^W ° " 200r 


1.6 


^^^\ o = 300r 


1.4 


^^. 


L2 


^^^^ 


1.0 


^^^ 


0.8 


\^ 


0.6 





10 
AGE (DAYS) 



20 



Fig. 4. Decrease in nucleocytoplasinic ratio between 1st and 20th postnatal days. 



values for neuron soma volume (nucleus + parikaryon ) in the 100 and 200 
r series are lower than in the controls in the first three developmental stages 
and increase to values above the control level on the 20th day. The group 
receiving the highest dose of irradiation (300 r) diverged more markedly 
from control values than the animals given lower doses, but the diflferences 
are within the range of error of the methods employed and are not statisti- 
cally significant. The nucleocytoplasmic ratio (Fig. 4), reflecting the chang- 
ing relationships between nuclear and cytoplasmic (perikaryon) volume 
through the four stages of development, was seen to decrease steadily in all 
groups with no significant differences appearing among irradiated or control 
groups. 

In contrast to the above findings, marked differences were observed in 
cortical thickness in area 2 (Fig. 5. Table I) between the controls and 
animals irradiated at 200 and 300 r {p < .01), and between the 200 and 
300 r groups themselves {p<.0\). No significant differences were noted 
between the 100 r groups and nonirradiated rats. 



FETAL X-RAY EFFECTS: CEREBRAL CORTEX 



33 



1600 



1400 



1200 



< 800 
o 

CE 
O 

o 60d 



400 




I - CONTROL 
t. = lOOr 
D = 200r 
o = 300r 



AGE (DAYS) 



20 



Fig. 5. Comparison of cortical thickness in area 2 in irradiated and control groups 
in early postnatal period. 

It is particiilaily noteworthy that the cufves illustratin<i the increase in 
cortical thickness for radiated and control ijroups tend to be parallel. While 
only the curves for area 2 are illustrated (Fiij. 5). it was observed on plottinti 
out the \alues for the other areas that the curves for each area are quite 
characteristic for that area, with the cunes for all radiated and control 
series showina: the same sjeneral trends. One notable exception to this rule 
was observed in area 4 in the 200 r series. Here the value for cortical thick- 
ness in the 200 r jjroup drops below that for the 300 r animals. With this 
exception, the differences in cortical thickness in area 4, 41, and 17 are of 
about the same order as in area 2. 

While cortical thickness thus appeared to exhibit an inverse relationship 
to radiation dose, no marked diflPerences in relati\e thickness of layers were 
observed in area 2 between irradiated and nonirradiated rats even in the 
300 r series. No detailed obser\ations other than cortical thickness were made 
in areas 4, 41, and 17. No a;eneral cytolos^ic differences between neurons in 
irradiated and control animals were observed in area 2. and no abnormalities 
of blood vessels were found. It is of course recognized that the latter would 
not be well demonstrated in Nissl preparations. 



34 BRIZZEE, JACOBS, KHARETCHKO AND SHARP 

TABLE I 

Comparison of Effects of Fetal X-irradiation at Various 
Doses on Cortical Thickness 



Age 


















(days) 


Area 


Control 


100 r 




200 


r 




300 r 


1 


2 


744 ± 14 


800 ± 


54 


655 


+ 


11 


546 ± 14 




4 


690 ± 20 


778 ± 


57 


560 


+ 


16 


551 ± 85 




41 


416 ± 26 


467 ± 


27 


370 


+ 


8 


314 ± 8 




17 


445 ± 17 


486 ± 


12 


396 


± 


22 


340 ± 9 


5 


2 


1131 ± 16 


1191 ± 


15 


982 


+ 


29 


845 ± 48 




4 


1086 ± 23 


1227 ± 


25 


975 


+ 


66 


778 ± 68 




41 


700 ± 61 


706 ± 


9 


545 


+ 


30 


445 ± 29 




17 


695 ± 17 


675 ± 


14 


549 


± 


14 


458 ± 45 


10 


2 


1529 ± 39 


1500 ± 


90 


1177 


± 


46 


1015 ± 55 




4 


1592 ± 28 


1470 ± 


48 


908 


+ 


65 


1021 ± 69 




41 


861 ± 14 


881 ± 


35 


858 


± 


19 


632 ± 80 




17 


920 ± 29 


896 ± 


18 


854 


+ 


10 


624 ± 37 


20 


2 


1654 ± 62 


1551 ± 


9 


1370 


+ 


58 


1221 ± 42 




4 


1668 ± 40 


1655 ± 


37 


1296 


+ 


92 


1080 ± 60 




41 


945 ± 23 


936 ± 


6 


843 


+ 


40 


673 ± 32 




17 


1112 ± 25 


977 ± 


21 


835 


± 


46 


670 ± 31 



Diflferences in brain weight (Fig. 6) between the 200 and 300 r series and 
between these groups and the control animals were consistent and marked 
throughout the four developmental stages studied. Brain weight in the 100 r 
group, however, was higher in the 1- and 10-day animals and appreciably 
lower in the 10- and 20-day stages than in the controls. At the 10- and 20- 
day stages the differences in brain weight between all three radiated series 
and between each of these groups and the nonirradiated rats were significant 
at the .05 level or less. 

Results of behavioral studies are summarized in Tables II and III and in 
Fig. 7. Table II shows the mean, variance, and number of rats for each of 
the nine groups in the experiment on locomotor ability. The higher the 
mean, the better the performance. From Table II it is clear that any initial 
differences between the groups are overcome by 140 days. An analysis of 
variance of these data, summarized in Table II, showed that the irradiation 
(totaling 320 r) did result in a decrement of locomotor coordination on the 
parallel bars for young rats. 

In the water maze experiment, 73 Cr of the control rats and 82 ^r- of the 
half-body rats reached the criterion of one perfect trial by the twenty-first 
trial, whereas only 26% of the full-body group did so (X" = 15.5, 2 df, 



FETAL X-RAY EFFECTS: CEREBRAL CORTEX 



35 



1.2 



en 

< 



z 

<0.4 
cr 

CD 



0.2 




1= CONTROL 
A = lOOr 
a '- 200r 
o = 300r 



10 
AGE (DAYS) 



20 



Fig. 6. Comparison of total brain weight in irradiated and control animals in first 
20 postnatal days. 



TABLE II 

Maximum ^-Inch Gaps Successfully Negotiated 
BY All Groups 







Conditions of Irradiation 




Days 
tested 


Full- body 


Half-body 


Controls 


40 


X = 2.90 


X = 8.67 


X = 8.50 




(7'= 1.69 


a' = 7.02 


a-' = 3.50 




N= 11 


N= 12 


^=6 


90 


X = 3.6 


X = 6.9 


X = 6.17 




(7^ = 2.04 


<r= = 9.49 


<r' = 3.76 




N= 10 


N= 11 


N^=6 


140 


X = 6.09 


X=7.36 


>r=5.8 




ff^ = 3.10 


<t'= 12.85 


a' = 6.20 




N= 11 


N= 11 


N = 5 



36 



BRIZZEE, JACOBS, KHARETCHKO AND SHARP 

TABLE III 



Summary Table for the Analysis of Variance of the 
Date from the Locomotor Experiment 



Source 


df 


MS 


F 


P 


Condition of irradiation 


2 


107.61 


18.75 


.001 


Age at test 


2 


8.20 


1.42 


. — 


Irradiation x age at test 


4 


23.58 


4.11 


.01 


Individuals 


74 


5.74 


— 


— 



p =z .01). Figure 7 represents the mean number of errors plotted against 
trials. One can see that the full-body group did not learn as rapidly as did 
the control and half-body groups and that the control and half-body groups 
were very similar. An analysis of variance of these data and the equivalence 
of the curves for the nonirradiated and half-body groups suggests that 
(a) the full-body group learned significantly more slowly than the other two 
groups, and (b) the effects of irradiating the mother with the uterus shielded 
did not cause a decrement in the future maze learning ability of their fetuses. 
Quantitative histologic methods in 50-day animals from the groups sub- 
jected to the behavioral tests revealed essentially the same differences be- 
tween full-body (320 r) groups and controls as were observed in the 20-day 
animals of the .300 r group described previously. That is, the only significant 



Half-body 

Controls 

Full-body 




Fig. 7. The mean number of errors for each group across trials. The data points 
were computed on the assumption that once a rat reached criterion he would make 
no more errors on successive trials. 



FETAL X-RAY EFFECTS: CEREBRAL CORTEX 37 

diflferences appeared in cortical thickness and brain weights and were gen- 
erally of the same magnitude as reported for the 300 r series. The values for 
the half-body group did not differ significantly from controls in any of the 
parameters studied. 

Discussion 

Rugh (1959) has made the observation that loss of cells in the embryo or 
fetus due to irradiation may be obscured by the fact that the organism has 
remarkable powers of integrating the remaining undiflferentiated, undam- 
aged neurectoderm into a topographically normal but reduced whole or- 
ganism. Oiu' results appear to furnish considerable suppoi t for this \ iew in 
reference to cerebral cortex. 

While the curxes illustrating increase in cortical thickness are plotted only 
tor area 2 i Fig. 5), it is seen on plotting the \alues for the other areas that 
the pattern of increase in cortical thickness in all radiated and normal series 
is characteristic for each area. While cortical thickness \aries inversely with 
radiation dose at the 200 and 300 r lexels in all areas, the cinves illustrating 
increase in cortical thickness at all dose lexels and in coiitrol series tend to 
be parallel tor a given cortical area, lliis strong tendency toward parallel 
growth within a specific area, together with the lack of any truly significant 
volumetric or density changes, indicates that the surviving cells must possess 
essentially the same de\elopmental capabilities as the nonirradiated cells ot 
the control animals. 

The degree to which the orientation and pattern of branching ot cell 
processes may differ in irradiated and control groups at the dose and rate 
levels used remains to be determined. The cjuantitatixe technique developed 
by Sholl (1953) and Eayrs (1955) should be admirably suited to the solu- 
tion ot this problem. However, it appears clear from our determinations 
that, at the dose le\els employed, there are no significant differences in cell 
territory (computed from neuron packing density) in any of the irradiated 
or control groups. This indicates that, whether or not the cell processes 
may be altered morphologically, they must, on the average, occupy the same 
cortical \olume in irradiated groups as in control animals. 

Hicks (1959) has shown that rather specific, predictable nervous system 
anomalies may be produced with single exposures ot x-irradiation from 
about 150 to 200 r at any given time in the gestation period after the 9th 
day. Our results suggest that with fractionated doses between the 10th and 
17th gestational days it may be possible to produce animals with cerebral 
cortexes in which definite and predictable cellular deficits, rather than gross 
anomalies, exist in the absence of any \olumetric. density, or general cyto- 
logic changes in the cells present. From our obser\ations it appears that the 



38 BRIZZEE, JACOBS, KHARETCHKO AND SHARP 

cellular deficiencies are generally distributed through all cortical layers and 
that neurons and neuroglia are equally afTected. If this were not true the 
neuroglia /neuron index would reveal more consistent differences between 
control and irradiated animals than were observed. We believe that the 
high values for neuroglial density and the neuroglia/neuron index in the 300 
r group in 1-day animals may reflect an increase in microglial elements as a 
manifestation of the terminal phases of the reparative processes which follow 
irradiation damage in the cerebral cortex as described by Hicks (1957). 

The use of graded fractionated doses of x-irradiation during the latter half 
of the gestation period may offer a means for producing a series of animals 
with predictable, graded cellular cortical deficits. One might raise the objec- 
tion, however, that fractionated doses administered on several successive 
days may produce cellular deficits in many or all parts of the neuraxis. This 
would obxiously decrease the \alue of such preparations for the study of the 
effects of localized cortical cellular deficits on learning ability, locomotor 
function, or electrophysiologic phenomena. It seems probable that better 
localized cellular deficits without gross abnormalities might be produced by 
fractionated closes given within a more limited time than used in the present 
study at certain specific days in the gestational period as shown by Hicks in 
his timetables of radiation malformations. Such specimens should prove 
particularly useful in the field of experimental psychology, and investigations 
aimed at exploring this possibility are now in progress in our laboratory. 

It is interesting to note that Hicks (1959) foimd that single doses of 150 
to 200 r administered on the 16th day of gestation resulted in cortexes which 
were only one-half as thick as normal and bore little resemblance to the 
normal laminated 6-layerecl neocortex except in the lateral region. In our 
preparations, even at total fractionated doses of 300 and 320 r, while the 
cortical thickness in area 2 is reduced by approximately 309r, the laminated 
character of the cortex in rats 20 and 50 days of age is well preserved with 
surviving cells apparently cytologically normal, thus indicating that the sur- 
vi\ing cells manage to overcome the effects of the daily low-dose radiation 
exposure to a great extent, even where the cumulati\e dose is rather high. 
In earlier stages, cell layers are less well de\eloped in many irradiated ani- 
mals, especially in areas 17 and 41. 

Nurnberger and Gordon (1957) have recently called attention to the in- 
adequacy of the more commonly used referents, such as wet weight and dry 
weight, for evaluating chemical and presumably metabolic properties of tis- 
sues and emphasized the desirability of employing more meaningful refer- 
ents, such as cell density. We would like to emphasize the desirability of 
employing not only cell packing density, but additional referents, such as 
mean and total nuclear \olume, cytoplasmic volume, glial packing density, 
glia/neiuon index, dendritic territory, cell surface, and other such parame- 



FETAL X-RAY EFFECTS: CEREBRAL CORTEX 39 

tcrs as a frame of reference foi properly exaluatinti the chemical properties 
and metabolic acti\ity of nerxous tissues. We emphasize this point because 
of the increasiny interest in chemical and metabolic effects of irradiation. It 
is our belief that more significant results will be derixed from such in\e.stiga- 
tions if they are based on the cellular composition of the tissue, rather than 
on tlu' more commonly used biochemical referents. 

With reference to the beha\ioral studies, since the sur\i\in<; cells in our 
irradiated preparations in area 2 appear cytologically normal, it appears that 
the explanation of the observed deficits in learning ability must rest on the 
assumption that the sur\i\ino cells possess physiologic deficits not amenable 
to measurement with quantitati\e histologic or ordinary cytologic methods, 
or that the deficit is entirely or in part the result of the numerical deficit in 
cells, or both. It is of considerable interest to note. howe\ er. that the loco- 
motor deficit obserxcd in the youngest animals tested is largely cleared up in 
the oldest group. Lonu-tcrm studies are now in progress to determine if a 
similar phenomenon may occur with relerence to learning ability in older 
animals following fetal iiiadiation. 

Summary and Conclusions 

Exposuie to fiactionated doses of total-body x-irradiation at 100. 200. and 
300 r (60 r per minute) o\er a peiiod of 5 to 7 days alter the 10th day of 
gestation results in no significant difference in neuron packing density in 
area 2 in the first 20 days of postnatal life. Nemoglial packing density and 
the neuroglial neuron index are higher in the 300 r group in 1 -day-old 
animals than in controls, but at later stages the differences are not signifi- 
cant. Mean \alues for neuron soma \oknne in ]-. 5-. and 10-day-old irra- 
diated animals are consistently lower than in nonirradiated series, but are 
higher at the 20-day stage in irradiated than in the control animals. The 
differences are considered to be within the range of error of the methods 
employed. The nucleocytoplasmic ratio decreased steadily fiom the 1st to 
the 20th days, and no significant differences occurred between irradiated 
and control series. 

Brain weight \aried in\eisely with irradiation dose at the 10- and 20-day 
stages and in 200 and 300 r groups at the 1- and 5-day stages. Clortical 
thickness in the 100 r group was not significantly different from controls. In 
the 200 r and 300 r groups, however, cortical thickness varied inversely with 
dose le\el, and significant diflerences betvyeen the 200 and 300 r groups and 
between the irradiated and control groups occurred at the 20-day stages in 
all areas. It is concluded that the doses of fetal x-irradiation employed pro- 
duced no significant dilTerences in volumetric or density relationships or 
general cytoarchitecture between irradiated and control animals in area 2 
but resulted in a significant decrease in total number of cells in this zone. 



40 BRIZZEE, JACOBS, KHARETCHKO AND SHARP 

References 

Brizzee, K. R.. and Jacobs, L. A. 1959. Early postnatal changes in neuron packing 
density and volumetric relationships in the cerebral cortex of the white rat. 
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Brizzee, K. R., Jacobs. L. A., and Kharetchko, X. 1961. Effects of total body x- 
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Eayrs, J. T. 1955. The cerebral cortex of normal and hypothyroid rats. Acta Anat. 
25, 160-183. 

Friede, R. 1954. Der quantitative Anteil der Glia an der Cortexentwicklung. Acta 
Anat. 20, 290-296. 

Furchtgott, E., and Echols, M. 1958a. Acti\ity and emotionality in pre- and nconatally 
x-irradiated rats. /. Comp. Physiol. Psychol. 51, 541-545. 

Furchtgott, E., and Echols, M. 1958b. Locomotor coordination following pre- and 
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Hawkins, A., and Olszewski, J. 1957. Glia/nerve cell index for cortex of the whole. 
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Heron, W. T. 1930. The test-retest reliability of rat learning scores from the multiple 
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Hicks, S. P. 1954. The effects of ionizing radiation, certain hormones, radiomimetic 
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in the nervous system, eye, and mesenchyme of the mammalian embryo after radia- 
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Hicks, S. P., DAmato, C. J., and Lowe, M. J. 1959. llie development of the mam- 
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Krieg, W. J. S. 1946. Connections of cerebral cortex. I. The albino rat. A. Topogra- 
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Levinson, B. 1952. Effects of fetal irradiation on learning. /. Comp. Physiol. Psychol. 
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Nurnberger, J. I., and Gordon, M. W. 1957. The cell density of neural tissues: direct 
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Rugh, R. 1959. Vertebrate radiobiology (embryology). Ann. Rev. Nuclear Sci. 9, 493- 
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Scholl, D. A. 1953. Dendritic organization in the neurons of the \isual motor cortices 
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ordination of albino rats. /. Comp. Physiol. Psychol., in press. 



structural and Behavioral Alteration in the 
Rat Following Cumulative Exposure of the 
Central Nervous System to X-lrradiation * 



Robert H. Brownson 

Medical College of Virginia, 
Richmond. J'irginia 



Introduction 

This project is constructed to permit analysis of the cytologic, histochenii- 
cal. and functional state of the rat's central nervous system. This analysis 
has been based on short- and lono-term responses demonstrated by the 
central nervous system to cumulative levels of total-head x-irradiation. The 
followins,' report is limited to the lonti-term responses. 

Da\ idofT and others !l938) have described the microscopic effects of x- 
irradiation applied directly to the brain and spinal cord of monkeys. These 
workers described thickeninu of the blood \essel walls, swelling", gliosis, 
hypertrophy, and formation of gitter cells in the nemoglial elements. These 
changes were noted 322 days following exposure in animals receiving 1,856 r. 
The same authors expressed the opinion that there seemed to be two factors 
involved in go\erning histologic alterations: the dosage and the time inter\al 
between irradiation and autopsy. 

Clemente and Hoist i 1954) subjected monkey heads to doses of x-irradi- 
ation ranging from 6.000 r to 15 r at 188 r per minute. They observed 
varying degrees of necrobiotic changes such as clumping, chromatolysis. 
degeneration, and neuronophagia of neurons with only slight changes be- 
low the 1,500 r le\el. These investigators obser\ed gliosis 4 to 8 months after 
the date of exposure. Astrocytes were hyperchromatic and inidergoing 
degeneration, while scattered oligodendroglia demonstrated swelling. 

More recently Berg and Lindgren il958i attempted to chart the extent 
of cerebral lesions produced by different divided and undivided doses and 
to determine the time-dose relationships for delayed reactions of brain tissue 
on application of a single dose and fractionated doses. These in\estigators 
reported \arious lesions in the rabbit brain : frank necrosis, partial destruc- 

* Supported by National Institutes of Health. 

41 



42 ROBERT H. BROWNSON 

tion of gray and white matter, as well as profound gliovascular alterations. 
The authors believe that their morphologic analysis supports the assumption 
that the vascular changes were primary and delayed; lesions in the brain 
weie essentially the same type after fractionated as single dose exposure. 

Materials and Methods 

In this study approximately 120 male 9-month-old rats were used. Of 
these, 54 were placed in a group for long-term studies whose postirradiation 
sacrifice dates were selectively extended. These animals were grouped in six 
large cages, 9 rats per cage. Dining the first week of x-irradiation, a total of 
36 animals were exposed; at random, 6 animals per cage received 1,000 r 
total-head irradiation. The remaining 18 animals, 3 in each cage, were 
maintained as controls. The rats received 1,000 r until the total accumulated 
dosage of the last remaining experimental group had reached 5,000 r. 

Control animals and experimental animals scheduled for radiation were 
fed during a 45-minute period each day for 6 days. On the 7th day, each 
animal was placed in a Skinner box for 45 minutes. The animals' first 
experience in the box resulted in learning to press a bar for food reward. 
For the next two periods in the box, the animals were placed on short 
aperiodic reward schedules. The third time, all animals were placed on the 
aperiodic schedules and bar presses recorded during a 45-minute interval. 
After establishing a desirable level of indi\idual performance, all of the 
\arious groups of animals underwent the scheduled exposure to x-irradiation 
and 5 weeks of testing. 

The head of each experimental animal was exposed to x-irradiation from 
a 1,000 kvp x-ray imit filtered through 2 mm of aluminum and 56 cm of 
air. A Victoreen Chamber R-Meter was used at each operation to obtain 
equal and exact data of the total roentgens delivered in a prescribed 
interval of time. The rats body was protected in a lead-lined box which 
was it.self shielded by a wall of lead bricks 2-3 in. thick from which the 
animal's unshielded head protruded vertically above the body shield. Each 
unanesthetized animal was secured in the box by means of a modified 
burette clamp that formed a collar and limited the animal's movement. 
The lead-lined box containing the rat was rotated clockwise through 360° 
at 33 rpm by a turntable. Each rat's head was continuously exposed to 
x-irradiation from multiple angles until the animal accumulated at one 
exposure a level of 1,000 r, delivered at the rate of 237 r per minute and 
J/a r per minute to a partially exposed neck. Each animal remained approxi- 
mately 4 minutes in the radiation chamber. As previously mentioned, this 
procedure was repeated every 7 days on the selected group of animals until 
the desired cumulative dosage was attained. The animal groups were then 



X-RAY INDUCED CNS CHANGES 43 

sacrificed after receivins; a total cumulative dosaoe of x-irradiation as 
follows: Group A, 1,820 r; Group B, 2,000 r: Group C:. 3,000 r; Group D, 
4,000 r; Group G. 5,000 r; and Group F, 5.000 r. 

All animals, control and experimental, were anesthetized with sodium 
nembutal and surs;ically prepared for perfusion \ia the left \entricle ot the 
heart. Perfusion pressure was maintained at approximately 90 mm of Ha;. 
Initially, the major portion of the circulatint; blood volume was removed 
by washina: the cardiovascular system with physiologic saline. A small in- 
cision in the right atrium provided a means ot exit for the perfused fluids 
and blood. Subsequently, with the heart still pidsatins;, the fixatives were 
perfused. Three animals received saline-acacia formalin, while the fourth 
animal received acetic acid-alcohol formalin. The partially fixed brain was 
then rapidly removed and cut by coronal sections into three parts: a frontal, 
midcoronal, and occipital plus brain stem and cerebellum. Each of the sec- 
tioned brains was then immersed in its respective fixative, formalin and 
acetic acid-alcohol formalin alona, with the control for each fi.xative. 

Following the usual histologic techniques, specimens fixed in acetic acid- 
alcohol lormalin were processed for histochemical analysis utilizing the 
periodic acid-Schiff" reaction (PAS) for olycogen. Specimens that were 
fixed in formalin-acacia were stained with toluidine blue for Nissl material 
and glia, azocarmine and VerhoefTs procedure for connective tissue and 
interstitial cell reaction, WeiTs method for general identity of structures 
and axon pathways, Swank-Davenport modification of Marchi method for 
degeneration of myelin, and Bodian's method for nerve fibers and nerve 
endings. 

Results 

Following each exposure to x-irradiation the animals showed behavioral 
changes which were unremarkable and somewhat variable. The animals in 
general exhibited confusion, sluggishness, or malaise, as expressed by with- 
drawal to the back of the cage. Generally, during the 24 to 72 hours follow- 
ing exposure, most animals appeared to recover from a postirradiation 
malaise. 

Group A animals (Table I and Fig. 12) survived 228 days after receiving 
a single exposure of 1,820 r and exhibited little or nothing in the way of 
physical changes. There occurred some slight weight loss during the first 
week which appeared to return to normal by the third week. One animal 
developed a head tumor, a benign involvement of the salivaiy glands, well 
encapsulated with a caseous center (sclerosing angioma). 

Group B animals (Table I and Fig. 12) received two successive exposures 
of x-ray totaling 2.000 r. The animals in this group averaged approximatelv 



44 ROBERT H. BROWNSON 

a \2^c decrease in body weisjht by the 228th day after initial exposure. 
These animals all demonstrated thinnina or diffuse loss of hair about the 
head with complete epilation around the eyes. All had \vell-de\eloped 
bilateral, posterior subcapsular cataracts. One animal developed a tumor of 
the head, a low grade carcinoma of the skin. 

Group C animals (Table I and Fig. 12) received a total of 3,000 r during 
3 weeks and were sacrificed 165 days after the initial exposure. The average 
decline in body weight over this time indicated approximately a 17 ^c loss. 
These animals all demonstrated a rather severe diffuse loss of hair about 
the head with complete epilation aroimd both eyes. 

Group D animals (Table I and Fig. 12) accumulated a total dosage of 
4,000 r during 4 weeks. Three of the animals were sacrificed after 158 days 
and demonstrated an approximate 20*"^ weight loss. 7\vo of the animals 
died after 44 days and demonstrated a loss of 319f of body weight. All 
animals showed diffuse loss of hair over the head with complete epilation 
around the eyes. 

Group G animals (Table I) accumulated 5,000 r during 5 weeks. Two 
animals were sacrificed after 116 days and demonstrated approximately a 
29% loss of body weight. All animals demonstrated severe, diffuse loss of 
hair over the head region with complete epilation around the eyes. 

The animals indicated thus far. in experimental groups A, B, C, D, and 
G along with their controls, ha\'e been utilized in behavioral studies during 
the first 5 weeks following radiation. The weight loss demonstrated by these 
animals, including controls, during this testing procedure was in part due 
to forced food deprivation. The calculations for weight loss were based on 
per cent of difference between controls and experimentals which allows for 
the standard weight reduction due to a decrease in rations. 

Certain animals died or were sacrificed as they became moribund, espe- 
cially in the 4,000 and 5,000 r dose range. Apparently this high mortality 
rate in these groups after total head irradiation is more inxolved than ap- 
parent. One additional group of animals, group F, recei\ed the same treat- 
ment as did group G, with the exception that these animals were not utilized 
in behavior studies and did not imdergo food deprivation. None of the 
animals in this group expired or appeared moribund before the date of 
sacrifice 228 days after initial exposure. All animals in this group had 
well-developed bilateral posterior subcapsular cataracts and the usual epi- 
lation. 

All groups of animals with the exception of group F underwent a pre- 
liminary analysis of behavioral characteristics in operant conditioning 
equipment. Results from a preliminary analysis of animal behavior indicated 
that, from the first day of exposine to x-irradiation, all groups of animals 
through and including the 4,000 r group demonstrated some decrease in 



X-RAY INDUCED CXS CHANGES 45 



TABLE I 



Cumulative Effects of X-irradiation 
Physical Findings'' 





Head 

dosage 

r" 


Weights 
(gm) 


PRT' 

(days) 


Hair 

loss 


Head 






Bleph- 
aritis Cataract 




Rat 


Con Exp 


Tumor 


(A) 


(B) 


(C) (D) 


(E) 


(F) 


(G) (H) 


(I) 



91" 





359 
















. 


98 





479 













. 





92 


1820 




580 


228 


— 


— 


+ 





93 


1820 




545 


228 





— 




+" 


94 


1820 




440 


228 


. — 


— 


— 




95 


1820 




460 


228 


— 


— 


. 


— 


96 


1820 




405 


228 


— 


— 


— 


— 


97 


1820 




460 


228 


— 


— 


— 


— 


99 





600 










. 








106 





490 







— 


— . 


— 


— 


100 


2000 




470 


228 


+ 


— 


+ 


— 


101 


2000 




424 


228 


+ 


— 


+ 


+ ' 


102'' 


2000 




292 


36 


+ 


+ 


+ 


— 


103 


2000 




475 


228 


+ 




+ 


— . 


104 


2000 




400 


228 


+ 


— 


+ 


— 


105 


2000 




475 


228 


4- 


— 


+ 


— 


107 





460 







— 


. 








114 





442 







— 


— 


— 


— 


108 


3000 




400 


165 


+ 


+ 


— 


— 


109 


3000 




368 


165 


+ 


+ 


— . 


— 


nil 


3000 




387 


165 


+ 


+ 


— 


— 


ill 


3000 




375 


165 


+ 


+ 


— 


— 


112 


3000 




406 


165 


+ 


+ 


— 


— 


113 


3000 




446 


165 


+ 


+ 


— 


— 


115 





429 







— 


— 


— 


— 


122 





416 




— . — 


— 


— 


— 


— 


116 


4000 




299 


158 


+ 


+ 


— 


— 


117 


4000 




317 


158 


+ 


+ 


— 


— ■ 


118" 


4000 




173 


115 


+ 


+ 


— 


— 


119 


4000 




390 


158 


+ 


+ 


— 


— 


120" 


4000 




303 


44 


-f 


+ 


— • 


— 


121" 


4000 




278 


44 


+ 


+ 


— 


— 


123 





378 







— 


— 


— 


— 


130 





369 







— 


— 


— 


— 


124" 


5000 




318 


44 


+ 


+ 


— 


— 


125 


5000 




336 


158 


+ 


+ 


— 


— 


126" 


5000 




292 


44 


+ 


+ 


— 


— 


127" 


5000 




324 


44 


+ 


+ 


— 


— 


128 


5000 




286 


116 


+ 


+ 


— 


— 


129 


5000 




241 


116 


+ 


+ 


— 


— 


■■^46 













— 


— 


— 


— 


F49 





— 







— 


— 


— 


— 


F48 


5000 




— 


'^o 


+ 


4- 


+ 


— 


F47 


5000 




— 


228 


+ 


+ 


+ 


— 


F50 


5000 




— • 


228 


+ 


+ 


+ 


— 


F51 


5000 




— 


228 


+ 


+ 


4- 


— 


F52 


5000 




— 


228 


+ 


+ 


4- 


— 


F54 


5000 




— 


228 


+ 


+ 


+ 


— 



•' Kky: '^1 pn-senci-: i — : absfiice: ' ' doi-s not applv. <i\io,ibuiid at sacrifice. 

■> Roentgens delivered in air. ' Benign encapsulated salivary gland (Sclerosing angioma). 

« Post-radiation time ' Low grade carcinoma. 

s .Animals not utilizfd foi behavioral studies; all otlui animals utilized in behavioral studies. 



46 



ROBERT H. BROWNSON 




90 120 150 

TIME IN DAYS 



240 



Fig. 1. Behavior reaction pattern demonstrated by control and experimental animals 
for food reward during 45 minute test periods. Slope of line to right indicates a gradual 
extinguishment of learned reaction. Bar [jresses are plotted against time in days. 



bar piessins^ acti\ ity for food reward (Fis^. 1). Those animals which re- 
cei\ed 1.000 r showed only minor deviation and after 228 days equaled the 
control animals. The 2,000 r animal group showed a more striking initial 
decrease but they. too. ecjualed the control animal performance by 228 days. 
Animals receiving the accumulated doses of 3,000 and 4,000 r both demon- 
strated a severe decrease in activity which reached the lowest performance 
rates 7 days after receixing their last exposmes. Dinging the remaining sur- 
vival period the 3,000 and 4,000 groups demonstrated little tendency to 
increase from this low level acti\ ity. The obvious decline of all animal 
performance o\ er the 5 weeks of testing, including controls, was attributed 
to gradual extinguishment of learned behavior. 

Purkinje cells and granule cells of the cerebellum appeared to have imder- 
gone certain similar changes at the various le\els of exposiue and time 
intervals. These changes appear to have in\oIved a mild to se\ere loss of 
Purkinje and granule cells. Hyperchromatic neiuons and pyknosis were 
prevalent throughout most exposure levels. These alterations (Figs. 2 and 3) 
were scattered throughout the cerebellum. 

Cerebral neurons demonstrated pyknosis and hyperchromatosis in scat- 
tered areas throughout all le\els of radiation. Mild neurofibrillarv or axonal 



X-RAY INDUCED C\S CHANGES 47 

changes, characterized by beading and swelling, were noted in all groups 
I Fig. 4). Occasional areas of cell necrosis were observed in the 5.000 r level. 

Hypothalamic neurons demonstrated occasional evidence of swelling, 
chromatolysis, pyknosis, and hyperchromatic staining qualities, but these 
changes were largely inconsistent. The presence of PAS-positi\e globules 
within the extraneuronal tissue was clearly evident over the entire dosage 
range of radiation, demonstrating no specific alteration. 

Brain stem neurons were not observed to ha\e undergone major changes 
at any level of radiation. Those changes that were obser\ed were mild to 
moderate chromatolysis. pyknosis. and hyperchromatism. In the 5.000 r 
level maintained 228 days, hyperchromatic neurons were in excess with no 
chromatolysis observed. 

The neuroglial cells underwent slight but consistent degrees of scattered 
hyperplasia in the subpial cortex ( Fig. 5 1 . These changes were somewhat 
localized and similar at the 1,820, 2,000, and 3,000 r levels. Astrocytes seem 
to have been the most regularly reactive cells and in inany instances could 
be termed gemestocytic. Glial cell hyperplasia and hypertrophy became quite 
noticeable in the cortex at the 4,000 and 5,000 r level, especially in the 
group surviving 228 days. Small areas of infarction (Fig. 6) were in 
the cortex and subcoitical white matter in all groups of animals. There 
seems to have been a more intensive cellular and fibrillary gliosis about 
these areas in the 4,000 and 5,000 r animals. 

The ependymal and subependymal cells were significantly reactive at all 
levels of radiation. There appeared to ha\e been a thinning of cells in all 
groups. The pyknosis and hyperchromatic cells were scattered throughout 
the varied exposuie le\els in different \entricular areas as well as within 
the same areas. It was not uncommon to find nests of hyperchromatic 
subependymal cell foci i Fig. 7). P.\S-positive accumulations of globules 
both within the basal position of the ependymal cell and subependymal 
areas were increasingly evident as the le\ el ot radiation was increased. Heavy 
deposits of such materials were noted in the 4.000 and 5,000 r groups. 

Meninges, specifically the leptomeninges. and the pial-glial membranes 
underwent changes Fig. 5i. \arying from mild thickening at 1,820 and 
2.000 r to moderately severe in the 5.000 r range. Such changes were 
noted to have been scattered o\er the brain in local areas. Throughout 
all of the \aried dosage levels occasional cellular and fibrillary infiltrations 
were present in these foci. .Astrocytes were gemestocytic in such areas along 
the piaglial membrane. 

Blood vessels underwent \arious alterations at the different dosage le\els. 
Vessels most often in\ol\ ed were capillaries and small arteries. Mild to mod- 
erate hypertrophy and hyperplasia of endothelium and increase in adventitia 
were noted in animals receiving 1.820, 2,000 and 3.000 r (Fig. 8) and were 



48 



ROBERT H. BROWNSON 



Iff' -^**Sa. 'tf*^^'^*^!^# 



> * 



.2 






I ^ 



if 



V / 




ti 




• 



X-RAY INDUCED CNS CHANGES 49 

severe at 5,000 r. Some petechial hemorrhages were seen. The animals that 
underwent 4.000 r exposure showed a more severe reaction. Apparently 
there was a consistent increase in connective tissue, presumably peri\ascular, 
from mild to se\ere within the hypothalamus from 1.820 to 5.000 r (Figs. 9, 
10. and 11 j. 

Discussion 

There were no remarkable or otherwise consistently observable neurologic 
deficits in animal activity during the 228 days following irradation. If any 
such alteration could be detected by observation alone, one might describe 
malaise. 

The animals" physical appearances were altered by cataracts and varying 
degrees of hair loss about the head. There was complete epilation of hair 
immediately surrounding the eyes in a circumscribing area approximately 
3 mm wide. In most instances the hair o\er the remaining portion of the 
rat's body lost its usual healthy sheen and became ruffled. Some of the 
animals had inflamed margins of the eyes which might be described as 
blepharitis. 

Analysis of weights indicated that beginning with group B at 2,000 r le\ el 
there was a decline in animal body weight. This was in the approximate 
magnitude of I2''r of control animal weight at the time of sacrifice 
228 days after initial exposure. Additional cumulative exposure to x-ray 
revealed a similar weight loss in the animals at sacrifice. The per cent of 
difference between the remaining groups of control and experimental animal 
weights became increasingly greater with increasing dosage. The greatest 
factor of difTerence was at the 5.000 r level where a loss of 29'^r was noted. 
However, the percentage weight differences indicated were taken only from 
weight differences at the time of sacrifice for the group as an a\"erage. In 



Fig. 2. Cerebellar granule cell pyknosis and hyperchromatic staining reaction 228 
days following 5,000 r. NissI: X 320. 

Fig. 3. Cerebellar granule cells demonstrating normal staining reaction. Nissl; 
X 320. 

Fig. 4. Cerebral corte.x focal area of pyknotic and hyperchromatic stained neurons 
228 days following 5,000 r. Nissl; X 130. 

Fig. 5. Cerebral cortex cellular gliosis and thickened pial-glial membrane with 
some cellular and fibrous infiltration of subarachnoid space 228 days following 5.000 r. 
Hematoxylin and eosin ; X 130. 

Fig. 6. Cerebral cortical infarct. Small with slight encapsulation of necrotic area 
228 days following 1.820 r. Verhoeff; X 320. 

Fig. 7. Ependymal cells and area of subependymal cell pyknosis and hyperchromatic 
staining reaction 228 days following 5,000 r. Nissl; X 320. 



50 



ROBERT H. BROWNSON 



'<'• 







10 





m&^^^ 










Fig. 8. Cerebral cortical arteriole demonstrating hypertrophy and hyperplasia of ad- 
vcntitia with large hyperchroniatic nuclei 228 days following 1.820 r. VerhoeflP: 
X 64. 

Fig. 9. Normal rat hypothalamus and third \entricle. VerhoefF; X 130. 

Fig. 10. Hypothalamus with increased vascularity and perivascular connective tissue. 
Increased numbers of nuclei 228 days following 2,000 r. VerhoeflF; X 130. 

Fig. 11. Hypothalamus with increased vascularity and perivascular connective tis- 
sue. Numbers of nuclei present less than demonstrated in Fig. 9 228 days following 
5,000 r. VerhoeflP: X 130. 

nearly all cases there was a favorable increase in the \veia;hts approximately 
one week followino; the last exposure administered to that group. One 
notable exception was in oioup G after reaching the 5,000 r level. This 
group failed to show any tendency to increase weight imtil after 120 days. 



X-RAY INDUCED CNS CHANGES 



51 



800 

700 

600 

500 
E 
J. 400 

5 300 

200 

100 



WEIGHT ANALYSIS 



Total Cumulative Dose 

• — • A 1820 r 

K « B 2000 r 

o C 3000 r 

» * D 4000 r 



Control 




ttt t 

ABC D Radiation Exposure (r) 



J I 



30 



60 



90 120 150 

TIME IN DAYS 



180 



210 



240 



Fig. 12. .Analysis ot average animal weights in grams for control and Lxperimental 
animals plotted against time in days. 



In no instance were the experimental animals in s,roups B. C, and D capable 
of equaling their control litter mates" weight record dininsj the 228-day 
interval before sacrifice. Animals in the control and e.xperimental cjroups 
underwent 5 weeks of behavioral studies which recjuired a substantial de- 
crease in food intake durin? observation. Dininq this period of food dep- 
rivation the control animal weights continued to rise (Fig. 12). empha- 
sizing that the decrease in body weight and life span of the rat following 
x-irradiation ot the central nervous system is further magnified bv forced 
reduction in available nutritional requirements. 

Cumulatixe exposure ol the rat head to x-irradiation has been noted to 
cause certain physical and histologic alterations. In the total accumulated 
dosages 1,820, 2.000. 3.000, 4.000 and 5.000 r these alterations were strik- 
ingly similar in character and appeared to show a Cjuantitative relationship 
to time and accumulated dose level. 

The cell changes, although quite similar in appearance, did display cei tain 
specific sensitivity. For example, in the cerebellimi both Purkinje and 
granule cell neurons underwent the severest reactions in rats receiving 
5.000 r after 1 16 days. This may be contrasted to those lesser cellular changes 
that were moderate in rats recei\ing 5.000 r and sur\i\in<; 228 days. It is 
possible that the inter\ening time between 1 16 and 228 days was sufficient to 
allow cell recovery. The cytologic changes were noticeably more severe in 



52 



ROBERT H. BROWNSON 

TABLE II 

Cumulative Effects of X-irradiation 



Physical data 



Microscopic 



Neurons 
Head 
Rats dosage PRT Purkinje Granule Pyramidal Tuber 

no. (r)"^ (days) cerebellum cerebellum cortex hypothalamus 



Large 
brain stem 



;a) (b) (C) 



(D) 



(E) 



(f; 



(g; 



(H) 



1820 228 Mild hyper- 
chromatic 



Mild scat- 
tered 
pyknotic, 
hyper- 
chromatic 



Scattered 
hyper- 
chromatic, 
pyknotic, 
mild neuro- 
fibrillary 
changes 



Mild to mod- — 
erate, (PAS) 
positive 
globules, 
mild hyper- 
chromatic 
pyknotic 



2000 228 Mild hyper- 
chromatic, 
pyknotic 



Mild scat- 
tered 
pyknotic, 
hyper- 
chromatic 



Scattered 
pyknotic, 
mild neuro- 
fibrillary 
beading, 
hyper- 
chromatic 



Heavy, (PAS) 
positive 
globules, 
hyper- 
chromatic, 
pyknotic 



Mild 

chromatolysis, 
hyper- 
chromatic, 
pyknotic 



3000 165 Mild hyper- 
chromatic, 
pyknotic 



Moderate 
scattered 
pyknotic, 
hyper- 
chromatic 



Scattered 
pyknotic, 
mild neuro- 
fibrillary 
brading 
hyper- 
chromatic 



Moderate Mild 
(PAS) posi- chromatolysis, 
tive globules, hyper- 
mild pyknotic, chromatic, 
hyper- pyknotic 
chromatic 



4000 158 Mild hyper- Moderate 
chromatic, scattered 
pyknotic pyknotic 

hyper- 
chromatic 



Scattered Moderate 
pyknotic, (P.^S) positive 

mild, neuro- globules 



fibrillary 
beading, 
hyper- 
chromatic 



moderate 
hyper- 
chromatic, 
pyknotic 



Moderate 
chromatolysis, 
hyper- 
chromatic, 
pyknotic 



5000 116 Severe Severe Scattered Heavy, (PAS) Moderate 

hyper- scattered mild neuro- positive chromatolysis, 

chromatic, pyknotic, fibrillary globules, hyper- 

pyknotic hyper- beading, hyper- chromatic, 

chromatic cell chromatic, pyknotic 

necrosis pyknotic 



5000 228 



Moderate 
to severe 
hyper- 
chromatic, 
pyknotic 



Mild 
scattered 
pyknotic 
hyper- 
chromatic 



Scattered Heavy, (PAS) Moderate 

pyknotic, positive to mild, 

mild neuro- globules, hyper- 

fibrillary hyper- chromatic, 

beading, chromatic, pyknotic 

cell necrosis pyl-^notic 



" Roentgens delivered in air. 
'' Animals utilized in behavioral studies. 
<• Animals not utilized in behavioral studies. 



X-RAY INDUCED CNS CHANGES 

TABLE II (Continued) 
CiMULATivE Effects of X-irradiatiox 



53 



Microscopic 



Glia 



Oligo- Astro- MicTo- 



Non-neural 



Ependyma 

sub- 
ependyma 



Meninges 



Blood 

vessels 



(I) 



(J) 



(k; 



(L) 



:mi 



(N) 



Scattered areas of Mild Mild 

cellular gliosis in pyknotic, scattered 

sub-pial cortex (PAS) thickening, 

gemestocytic astrocytes. positi\e cellular 

occasional small infarct globules infiltration 



Moderate increase in 
perivascular connective 
tissue and blood vessels in 
hypothalamus, hypertrophy, 
hyperplasia of endothelium 
and ad\entitia 



Occasional small Heavy Mild Moderate increase in 

infarct scattered (PAS) scattered perivascular connecti\e 

cellular gliosis in positive thickening, tissue and blood vessels in 

sub-pial cortex globules cellular hypothalamus, hypertrophy, 

infiltration hyperplasia of endotheliimi 
and adventitia 



Scattered areas of 


Hyper- 


Mild 


cellular gliosis in 


chromatic, 


scattered 


sub-pial cortex 


pyknotic, 
(PAS) 
positive 
globules 


thickening 


Mild cellular and 


Heavy 


Mild 


fibrillary gliosis 


(PAS) 


scattered 


around infarcts in 


positl\-e 


thickening 


white matter 


globules 





Mild increase in 
peri\ascular connective 
tissue and blood vessels in 
hypothalamus, mild hyper- 
trophy and hyperplasia of 
endotheliiun and ad\'entitia 



Moderate increase in 
perivascular connective 
tissue and blood \-essels in 
hypothalamus, mild hyper- 
trophy and hyperplasia of 
endothelium and ad\entitia 



Mild cellular and Heavy Moderate 

fibrillary gliosis (P.AS) scattered 

around infarcts in positive thickening 

white matter globules 



Moderate increase in 
perivascular connective 
tissue and blood vessels in 
hypothalamus, moderate 
hypertrophy and hyperplasia of 
endothelium and adventitia 



Moderate cellular Heavy Moderate 

and fibrillary (PAS) scattered 

gliosis in sub-pial positive thickening, 

cortex, gemestocytic globules cellular 

astrocytes, small infiltration 

infarcts in white 
matter 



Heavy increase in 
perivascular connective 
tissue and blood vessels 
in hypothalamus, severe 
hypertrophy and hyperplasia of 
endothelium and adventitia 



54 ROBERT H. BROWNSON 

the granule cells than in Purkinje cells, appearing in both as nuclear pyknosis 
and hyperchromatic staining. There appeared to be an appreciable decrease 
in cellularity most severely affecting the 5,000 r level at 1 16 days. 

Wilson ( 1960) irradiated monkeys with whole-body exposure to cobalt-60 
(gamma) from 400 to 40,000 r and stressed the changes that occurred in 
animals dying during the first 54 hours post-irradiation. Cerebellar granule 
cells underwent nuclear pyknosis especially within the innermost layers 
with a decreased cellularity explained on the basis of decreased nuclear area. 
Vogel ( 1959) administered massive doses of gamma radiation to the head of 
rabbits and dogs sacrificed at intervals up to 10 days. He reported that the 
altered granule cells of the cerebellum demonstrated pyknotic and hyper- 
chromatic nuclei most notably within the first 24 hours after exposure. His 
opinion was that the recovery phase was completed by 72 hours after 
radiation. 

The tissues studied in this project are decidedly those of chronic classifi- 
cation sacrificed up to 228 days after exposure. The results from oiu' study 
indicate that x-irradiation of the rat head will elicit slight changes in cere- 
bellar neurons after 228 days and that a dose as high as 5,000 r is capable of 
causing severe cell change during the 1 16 days. In all instances the changes 
described through and including 228 days after radiation were accompanied 
by decreased cellularity. It would only be speculation to propose cell loss. 
The only absolute measure of this would ha\e to incorporate a quantitative 
analysis. 

Wilson (1960) and Vogel's (1959) findings that the granule cells in the 
cerebellum undergo acute changes followed by reco\ery during a slightly 
later period after radiation, would seem to indicate the existence of secondary 
effects. This effect noted months after exposure may further demonstrate 
its recovery phase at a still later date. 

Neurons in the cerebral cortex, hypothalamus, and brain stem seldom ap- 
peared to be necrotic and were probably of reversible nature. Such changes 
were described as shrinkage in both cytoplasm and karyoplasm or pyknosis. 
It is suspected that the decrease in cell or nuclear volume or both is para- 
mount to the hyperchromatic staining quality, as well as to the appearance 
of decreased cellularity. Peculiar to these alterations was the obvious scat- 
tering of cell changes within identical structiue, thus indicating certain 
differences in radiosensitivity within the particular structure imder observa- 
tion. In view of the perfusion-fixation methods utilized for this study, it is 
felt that these observations are reasonably free from artifact. These cell 
changes were present in varying amounts throughout all radiation levels, 
which seemingly indicates little or no direct qualitati\e relationship between 
dose and time within the parameters of this study. Only at the 5,000 r level 
were there evidences of significant neuronal necrosis. The absence of major 



X-RAY INDUCED CNS CHANGES 55 

structural alterations amon<j cortical neurons has been pointed out re- 
peatedly by many in\estioators ( Gerstner it al., 1956: Haymaker et al., 
1954, 1958: Russell vt al., 1949i for a wide ranse of experimental variables. 

Gliosis was evident in increasino quantity in direct relationship to the in- 
creasing) level of cumulated dosage or the postirradiation time or both. The 
sjliosis was characterized by increased cellularity and, for the most part, ap- 
peared within the molecular layer of the cortex, specifically the subpial 
area. The cell hyperplasia occurred predominantly in astrocytes, many of 
which appeared gemestocytic. 

Oligodendroglia and microglia remained relatixelv nonresponsi\e or 
negative. Increased fibrillary responses were noted only as thickenings or 
encapsulating responses about small areas of infarct. Arnold and Bailey 
I 1954) ha\e placed particular emphasis on glial response in the monkey 
brain to high and low energy x-rays. They reported that glial responses 
were related to dose intensity and time. High energy, low doses, e.g., 
3.000-5,000 r. initiated astrocytic responses as hypertrophy and hyperplasia 
months after exposure. Globus and others i 1952) implanted radon seeds in 
the brain stem of dogs and after 106 days reported the appearance of astro- 
cytosis. In an earlier study of glial response to 1.500 r x-irradiation in the 
guinea pig. Brownson ( 1960) found no significant alteration of either quali- 
tative or quantitati\e relationships of perineuronal satellite glial cells in 
various ages of animals 33 days after exposure. Glia have been reported (o 
demonstrate responses related to duiation of time after irradiation, that is, 
initially inhibition, then reco\ery. and eventually intense gliosis (Arnold 
and Bailey, 1954). 

Ependymal and subependymal cells lining the ventricular system appeared 
to demonstrate selectixe sensiti\ity. Rats. 7 days after exposure to cumulated 
doses between 1,000-5,000 r ot total head x-ray. frec]uently demonstrated 
ependymal and subependymal cell swelling and pyknosis (Brownson. 1961). 
Hicks and Montgomery i 1952) ha\e noted subependymal necrosis in rats 
6-12 hours following head exposure to 1,200 r. There were occasional scat- 
tered nuclear pyknosis of ependymal cells and nests of hyperchromatic 
subependymal cells in all le\els of .x-irradiation doses. Heavy accumulations 
of PAS-positive globules were present in all radiation groups. These dark 
globules were located within the basal portion of ependymal cells and sub- 
jacent extracellular areas and were not affected by previous treatment with 
salivary enzymes i diastase ) . 

C:hanges in the walls of blood \essels were regular throughout the tissues 
studied. These alterations predominantly appeared as hypertrophy and hy- 
perplasia of the ad\entitia, often with extra nuclei. Less obvious were 
changes in endothelium demonstrated as hypertrophy and hyperplasia. The 
total picture suggested that the dosage accumulated up to and including 



56 ROBERT H. BROWNSON 

5,000 r had little differential effect on blood vessels. Ho\ve\er, the added 
factor of 228 days in either low level or hiijh le\el dosa|2;e was critical, as 
evidenced by increased severity of reaction. 

Lyman and his colleagues ( 1933) , in an early study of the effects of roent- 
gen rays on the central nervous system of adult dogs, reported that 6 months 
after irradiation various sized blood vessels of the brain had progressive 
changes. These changes were quite similar to those seen in our animals, 
with marked hyaline degeneration and obliterating sclerosis of arterioles. 

In the hypothalamus of radiated animals there was especially heavy con- 
nective tissue, which might be thought of as an increased vascularity involv- 
ing pial-glial extensions from the surrounding meninges. Furthermore, 
there appeared to be an increased number of nuclei, mostly neuroglial. In- 
creases in cellularity in animals receiving 2,000 r at 228 days following ex- 
posure were less severe in animals which received 5,000 r after 228 days. 

Conclusions 

Rats were exposed to total head x-irradiation in doses of 1,000 r per week 
until accumulations by groups of six animals ranged from 1,820 to 5,000 r. 

During the inter\al following each exposure to ionizing rays, animals in 
all dosage levels demonstrated changes in behavior, physical appearance, 
and weight. Quantitati\e food reduction had a deleterious effect on the 
general well-being and sur\ival of the animals recei\ing the larger ac- 
cumulated doses of x-irradiation. 

Neiuons in the cerebral cortex, brain stem, and cerebellum demonstrated 
slight to severe alterations throughout all dose levels. Generally, cell change 
became more frequent and in some instances more severe with increasing 
accumulation of total roentgens and time following exposure. 

Neuroglial elements underwent variable minor alteration. Astrocytes were 
most frequently observed undergoing hyperplasia and hypertrophy. 

Meningeal thickening with some evidence of cellular and fibrillary infiltra- 
tion was observed throughout all dose levels. 

Endothelial and adventitial changes were noted in small and medium 
size intramedullary vessels with perivascular accumulations of large nu- 
cleated cells. Changes in blood vessels were directly related to increasing 
dosage and time. 

General obser\ation indicated that changes in cells leading to necrosis 
were similar throughout the various dose levels and days after exposure. The 
increasing severity in reactions associated with time and dose were more 
quantitative than qualitative. 



X-RAY INDUCED CXS CHANGES 57 

References 

Arnold, A., and Bailey. P. 1954. Alterations in the glial cells following irradiation of 

the brain in primates. A.M. A. Arch. Pathol. 57, 383-391. 
Berg, N. O., and Lindgren, M. 1958. Time-dose relationship and morphology of de- 
layed radiation lesions of the brain in labbits. Acta Radiol. Suppl. 167, 1-118. 
Brownson, R. H. 1960. The effects of x-irradiation on the perineuronal satellite cells 

in the cortex of aging brains. /. Neuropathol. Exptl. Neurol. 19, 407-414. 
Brownson, R. H. 1961. Changes inducted in the rat central nervous system following 

cumulative exposure to x-irradiation. .A. short term study. /. Neuropathol. Exptl. 

Neurol. 20, 206-218. 
Clemente, C. D., and Hoist. E. .\. 1954. Pathologic changes in neurons, neuroglia and 

blood-brain barrier induced by x-irradiation of heads of monkeys. A.Ai.A. Arch. 

Neurol. Psychiat. 77. 66-79. 
DavidofT, L. M., Dyke. C. G.. Elsberg. C. A., and Tarlov. I. M. 1938. The effects of 

radiation applied directly to the brain and spinal cord. Radiology 31. 451-463. 
Gerstner. H. B., Brooks, P. M., Vogel, F. S.. and Smith. S. A. 1956. Effect of head 

x-irradiation in rabbits on aortic blood pressure, brain water content, and cerebral 

histology. Radiation Research 5, 318-331. 
Globus, J. H., Wang, S. C, and Maibach, H. I. 1952. Radon implantation in the 

medulla oblongota of the dog: effects on the degree and extent of cellular reactions. 

/. Neuropathol. Exptl. Neurol. 11, 429-442. 
Haymaker, VV., Vogel, F. S., Cammermeyer, J., Nauta, W. J. H. 1954. Effects of high 

energy total-body (gamma) irradiation on the brain and pituitary gland of monkeys. 

Am. J. Clin. Pathol. 24. 70. 
Haymaker, VV., Laquer. G. L., Nauta, VV. J. H., Pickering, J. E., Sloper, J. G., and 

Vogel, F. S. 1958. The effects of barium 140-lanthanum 140 (gamma) radiation 

on the central nervous system and pituitary gland of Macaque monkeys. /. Neouro- 

pathol. Exptl. Neurol. 17, 12-57. 
Hicks, S. P.. and Montgomery, P. O'B. 1952. Effects of acute radiation on the adult 

mammalian central nei-\ous system. Proc. Soc. Exptl. Biol. Med. 80, 15-18. 
layman, R. S., Kupalow P. S.. and Scholtz, VV. 1933. Effects of roentgen rays on the 

central nervous system. A.M. A. Arch. Neurol. Psychiat. 29, 56-87. 
Russell, D. S.. Wilson. G. VV.. and Tansley, K. 1949. Experimental radio-necrosis of 

the brain in rabbits. /. Neurol. Neurosurg. Psychiat. 12. 187-195. 
Vogel, F. S. 1959. Changes in the fine stiucture of cerebellar neurons following ioniz- 
ing radiation. /. Neuropathol. Exptl. Neurol. 18, 580-589. 
Wilson, S. G. 1960. Radiation induced central ner\ous system death. /. Neuropathol. 

Exptl. Neurol. 19, 195-215. 



Behavioral and Histologic Effects of Head 
Irradiation in Newborn Rats 

James N. Vamazaki. Leslie R. Bennett, and Carmine D. Clemente 

Univeruty of California, Los Angeles Medical Center and \.A. Hospitals, 
Los Angeles and Sepulveda, California 

Introduction 

The efiect ot ioniziiiii radiation on the nei\ous systt-ni durinti \aiioiis 
stages of its o^rowth and de\eIopinent has elicited a broad spectrum of 
pathoio<_;ic responses. Early e.xperirnents clearly demonstrated the relative 
\ ulnerability to radium and x-ray of the brain in youni^ guinea pigs. rats, 
doys, rabbits, and kittens compared to the adult i Danysz. 1903: Turner 
and Geort;e. 1910). resultiny in stunted iirowth and abnorn:ial neurologic 
findings. The teratogenic effects of ionizing radiation in the embryo has 
received the attention of many excellent studies clarifying the differential 
response during the various stages of development from the earliest embry- 
onic stage to the end of gestation i Bagg. 1922: Job ct al.. 1935: VVarkany 
and Schraffenberger. 1947: Russell and Kussell. 1954: Hicks. 1950, 1953a.b. 
1954a,b). The nature of the cerebral anomalies depended less on the dose 
ot radiation than on the time of gestation when administered. The degree 
ot sensitivity has been related to the extieme sensitivity of the undifteren- 
tiated multi-potential cell in the \ery early embryonic stage and later in 
gestation to the early neural cell or neuroblast ( Rugh and Grupp. 1959: 
Hicks. 1950, 1953a.b. 1954a.b). 

Regardless of the ease with which the embryonic central nervous system 
can be damaged, an increasing gradient of radioresistance develops as gesta- 
tion progresses. The degree ot this sensitivity is demonstrated by the fact 
that 15 r can produce exencephalia in the very early embryo (Rugh and 
Grupp. 1959). In contrast, the minimal dose which has caused pathologic 
lesions in the adult monkeys has been 1.500 r when slight neinonal damage 
was seen, but some of the animals after 4 to 8 months had elapsed developed 
focal seizures. With larger doses there occurred blood brain barrier changes, 
astrocytic and neuronal damage with lesions, particularly affecting the hypo- 
thalamus and the medulla. Several neurologic signs of varying severity 
developed according to the radiation dose (Clemente and Hoist, 1954: 

59 



60 YAMAZAKI, BENNETT AND CLEMENTE 

Arnold et al, 1954a,b,c; Davidoff it ai, 1938). Many adult rats receiving 
1,000 r to the head alone appeared normal in every respect (Bennett, 1960) . 
In young head-irradiated rats of unspecified age, it has been reported that 
subependymal cells of the lateral \entricle were destroyed with 200 r. How- 
ever, the associated neurologic findings were not mentioned (Hicks and 
Montgomery, 1 952 ) . 

The time sequence dining which this radioresistance de\elops in the 
neonatal period associated with the maturation of the brain has not been 
as clearly demonstrated. In the fetal guinea pig, the neuronal differentiation 
is closely correlated with biochemic, functional, and other morphologic 
changes in the last trimester of gestation ( Flexner, 1953). The monograph 
edited by Waelsch (1955) emphasizes that in the rat similar developments 
occur during the first 10 postnatal days. Ionizing radiation inight well 
alter these highly integrated events suggesting the possibility of a changing 
pattern of response to irradiation with increasing age. Our study was under- 
taken to systematically observe the immediate and long term behavioral and 
histologic effects of single direct radiation graded doses to the brain of 
neonatal and young rats. 

Method 

A total of 112 rats, aged 8 hours to 15 days, recei\ed x-radiation to the 
head only. Single doses of radiation were given at four levels: 125, 300, 
500, and 1,000 r. Observations were made on 48 control animals. From 48 
to 72 hours after irradiation, 28 animals were autopsied. The remaining 84 
animals were observed up to 14 months and sacrificed as described in pre- 
vious reports (Yamazaki ct al., I960: C'lemente, ct ai, 1960). 

Observations 

The most interesting data in our study indicated that the severity ol the 
nein'ologic and pathologic findings were dependent on two factors: the 
intensity of the radiation dose and the stage of postnatal maturation of the 
brain at the time of radiation. The most severe brain reactions were found 
in rats radiated on the first 3 or 4 days postnatally. Animals irradiated on 
the 5th postnatal day definitely showed fewer neurologic signs than rats 
radiated on the first 4 days. In the 5-day rat, 500 r administered to the head 
produced neurologic signs in a little less than half of the irradiated rats, 
whereas all of the animals irradiated with 300 r and 500 r prior to this 
time developed some evidence of central nervous system involvement, and 
1,000 r often proved lethal during the first few days of life. Animals radiated 
after the 5th postnatal day showed a marked decrease in the incidence and 



EFFECTS OF HEAD IRRADIATION IN NEWBORN RATS 



61 



lOOOn 






800- 



600 



400- 



200- 



/ 



t- 



4 6 8 10 

DAYS AFTER BIRTH 



14-17 



Fig. 1. A graph showing a dose-age cur\c with respect to tlie production of neu- 
rologic signs by head radiation in the neonatal rat. Most of the animals to the left of 
the cur\e had clinical signs in\ol\ing the ner\ous system, while most of the animals 
to the right of the cur\e did not. (From CUcmente et a!.. 196(1. p. 670.) 



sexerity of neurologic signs, and radiation on the 15th day resulted in no 
visible neurolotiic findings. e\en thouy h doses of 1 .000 r were administered 
(Fi,o-. 1). 

Similar a^e-dose relationship was demonstrated in regard to mortality, 
retardation of body growth, head growth, and cataract formation. Tremor 
was the most frecjiiently obser\ed neurologic sign and appeared as early as 
the 2nd week in animals irradiated with 1.000 r during the first 5 days 
postnatally. Other neurologic findings were incoordination, paresis, a pe- 
culiar propulsixe gait, and falling backward when animals attempted to 
stand on their hind legs. Seizines occurred in a few animals. Small heads 
were noted in 23 cases. Cataracts dexeloped in 7 rats. 6 of whom had small 
heads, and the greatest incidence of cataract formation occurred in animals 
that had received 500 r on the 5th day. It is possible that a higher incidence 



62 YAMAZAKI, BENNETT AND CLEMENTE 

TABLE I 

Weight of Head-Irradiatkd Rats at 3 Months of Age 

No. of animals Range (gm) Average weight (gm) 
Male 



500 r 












Rx 1 - 


5 


days 


10 


170-280 


234 


Rx 6 - 


15 


days 


5 


255-290 


269 


1,000 r 












Rx 1 - 


5 


days 


9 


140-220 


180 


Rx 6 - 


15 


days 


4 


220-260 


246 










Female 




Controls 






17 


160-225 


185 


500 r 












Rx 1 - 


5 


days 


12 


125-185 


165 


Rx 6 - 


15 


days 


12 


170-220 


198 


1,000 r 












Rx 1 - 


5 


days 


2 


110-140 


125 


Rx 6 - 


15 


days 


6 


140-190 


175 



of eye patholo<iy niiiiht have been found in animals radiated under 4 days 
of as^e had they been able to survive for longer periods. 

Animals radiated during the first 5 days of life tended to weigh less than 
controls (Table I). Mortality was also greatest in the animals irradiated 
during this immediate neonatal period, and, with one exception, all deaths 
under 3 months of age occurred in this group. The average survival periods 
of the animals that died under 3 months of age were 0.9 months for the 
1.000 r animals, and 2.3 and 2.4 months for the 300 and 500 r animals, 



TABLE II 
Mortality in Head-Irradiated Rats 

Radiation dose Age at the time of irradiation 



(r) Under 4 days 5-6 days 7-15 days 

125 0/3 — — 

300 3/9 — 0/2 

500 5/13^' 5/17 0/3 

1,000 9/11" 1/2 2/14 

One anesthetic death. 
' Two anesthetic deaths. 



EFFECTS OF HEAD IRRADIATION IN NEWBORN RATS 63 

iesp(.'cti\ely. The difference in brain in\ol\ement between the animals radi- 
ated durino the first 5 days ot Hfe compared to the older animals is demon- 
stiated by the followino examples: 

500 r: Rat No. 32 was irradiated on the 5th day of life. Tremors were 
first noted at 6 weeks. These became progressively more severe, and in- 
coordination and weakness of the hind limbs were noted from the 3rd 
month. The eyes and head were small, and cataracts developed in one eye. 
Of 7 animals radiated after the 8th day, only rat No. 18 developed any 
neurolo2;ic siijn. a mild tremor first noted at the 10th month. 

1000 r: Animals ladiated on the first days of life at this dose level denron- 
strate the marked vulnerability of the brain at this age. By the 2nd week 
of life they could easily be separated from their control litter mates by their 
small size, weighing an average of 18 gm in comparison to 39 gm for the 
controls. Marked tremor was alieady present, eyes were narrowed and small, 
the animals tended to drag their hind limbs and encountered difficulty in 
righting themselves. Other animals revealed a marked hypeiactivity. darting 
about the cage in a purposeless, random manner. 

The incidence and severity of the pathologic findings correlated closely 
with the neurologic findings in the irradiated animals. 

The most consistent and outstanding finding was the difk-retwje in size of 
the cerebellum in the irradiated animal in comparison to the controls. When 
the cranial vault is lemoved in a newborn rat. the cerebellum is in close 
approximation to the inferior coUiculi ot the brain stem. Within a week 
after birth, the hemispheric colliculi are overgrown by the developing cere- 
bellum and cerebral cortex in the normal animal, whereas in the irradiated 
animals the colliculi are verv prominent even 3 weeks after birth. The 
ceiebellum remaining is underdeveloped. Petechial hemorrhages were noted 
on the surface of the cerebral cortex, although noticeably absent elsewhere. 
When the irradiated brain was cut. the tissue had a more gelatinous 
consistency than the normal brain. 

In animals allowed to survive lor longer periods of time, the generally 
retarded size of the brain was striking in addition to a distortion in the 
configuration of the cerebellum. The meninges often appeared thickened 
and fibrous, and the diua was firmly adherent to the brain surface, although 
it was possible to separate it from the cerebral cortex. The posterior part 
of the cortex often was depressed as if the cortex were thinner in this area. 

In the animals sacrificed 48 to 72 hours after irradiation, widespread 
changes in the vasculature in the brain and brain stem were noted, and 
more specific lesions were identified in the choroid plexus and meninges. 
Vascular lesions occmred after dosages of at least 500 r in animals irradiated 
within the first 5 days postnatally and in animals administered 1.000 r 
between the 5th and 10th postnatal days. The most common vascular 



64 



YAMAZAKI, BENNETT AND CLEMENTE 



phenomena observed 2 to 3 days after irradiation seemed to be simple 
swelling of the endothelial cell cytoplasm with a concomitant increase in 
nuclear basophilia (Fig. 2), petechial hemorrhages at the capillary level, 
and, in some instances, polymorphonuclear vasculitis. In the choroid 




EFFECTS OF HEAD IRRADIATION IN NEWBORN RATS 



65 



plexus, in addition to the capillary changes which were at times limited to 
the plexus in the fourth ventricle, the parenchymal cells appeared sw^ollen 
and cytoplasmic material more watery than normal, and the cells contained 
relatively little granidar material. Acute inflammatoiy reactions noted in 
the meninojes were obser\ed in most animals oiven over 300 r within the 
first 8 postnatal days (Fig. 3). Tht- meningitis, characterized by neutro- 




FiG. 3. (a) .\ photomicrograph showing, in trans\erse section, the \iiitral mid- 
pontile region in a 23-day-old rat that had been given 1000 r to the head on the first 
postnatal day. Notice the thickened meningeal coverings (arrow) and the thickened 
vascular channels of the pia mater. (X 32) (b) .\n enlargement of the block out- 
lined in a, showing more clearly the thickened \ ascular walls (anows). The lumen of 
the vessel on the right was almost completely obstructed. Thioninc stain (X 130) 
(From Clemente et al.. 1960. p. 670.) 



66 YAMAZAKI, BENNETT AND CLEMENTE 

philic accumulation in the dura and around the \a.sculature of the pia 
mater, tended to be more focal with the lower radiation dose and more 
intense and generalized with the higher radiation dosages (1,000 r). 

In the long term series (survival periods of 20 days to 14 months), 
vascular pathology consisted principally ol an increase in the thickness of 
vessel walls with a consequent narrowing of the vascular lumen. This was 
especially evident in vessels that were seen within the meningeal layers on 
the surfaces of the brain, although at times deeper vessels also showed im- 
mistakable signs of damage. The thickening in the blood vessel walls was 
most pronounced in the tunica media and in the tunica intima. so that the 
total vessel diameter did not increase, but the lumen decreased. Aroimd such 
fibrosed vessels there often were signs of a minor chronic inflammatory 
process. In these instances, the inflammatory reaction, characterized by the 
presence of mononuclear cells, was possibly the residual of the more acute 
inflammatory reactions observed in the short term series. 

Lesions observed over a longer time revealed hemorrhage and necrosis in 
areas where capillary damage was most e\ ident in the brain of animals 48 
to 72 hours after irradiation, suggesting that this initial lesion was an acute 
phase of the lesions observed at later periods. The areas that were most 
often involved included the cerebellum, basal ganglia (especially the globus 
pallidus and caudate nucleus), diencephalon, and medulla. In many rats, 
sections of the brain stem showed an excessive number of neuroglial cells, 
even though no distinct localized lesions could be seen. This hypergliosis 
unassociated with apparent neuronal damage was most intense in white 
matter and most frequent in the midbrain, pons, and medulla, but was also 
seen in the caudate nucleus and globus pallidus. 

Evidence of early inflammatory lesions in tlie choroid plexus was visible 
as small contracted scars characterized by connective tissue proliferation. 
Marked enlargement of the lateral ventricles developed in animals receiving 
1,000 r during the immediate neonatal period (Fig. 4). With 500 r the dis- 
tension of the ventricles was not as noticeable. The third and fourth ventri- 
cles were never as severely involved. With lateral ventricular distension, 
there occiared a concomitant decrease in thickness of the cortical layers. The 
packing density of the cortical neinons looked like that of the 3-day-old rat, 
although some of the animals had lived from 3 weeks to 3 months. The 
cortical neurons appeared intact; however, the cortical mantle resembled 
that of a much younger animal. 

The dura and pial coverings of the brain increased in thickness with time. 
Proliferation of connective tissue and a mild secondary mononuclear infil- 
tration occurred, especially in animals irradiated with 1,000 r between the 
5th and 14th day and surviving for 9 months to one year. With meningeal 



EFFECTS OF HEAD IRRADIATION IN NEWBORN RATS 




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68 YAMAZAKI, BENNETT AND CLEMENTE 

thickenino, there invariably was some thickening in the walls of the medium 
sized and smaller arteries of the pial layers. 

Discussion 

The neurologic findings observed in the rats in this study are similar to 
the findings of other investigators in their studies on irradiated newborn and 
young guinea pigs, rabbits, kittens, and dogs (Danysz, 1903: Turner and 
George. 1910; Brunner, 1920; Nemenov, 1934; Demel 1926; Mogilnitzky 
and Podljaschuk, 1930). Tremors, clonic twitching, epileptoid seizures, pa- 
ralysis of extremities, retardation of head and body size, and poor mental 
performance have been reported. Previously, however, a systematic sequen- 
tial age-dose relationship during the neonatal period had not been demon- 
strated. 

The eflfect of radiation on the developing human brain is not as well docu- 
mented as are the animal studies. The occurrence of microencephaly, mental 
defectives, hydrocephaly, ossification defects of the cranial bones, eye defects, 
and skeletal abnormalities in children born after maternal pelvic irradiation 
has been reported (Murphy, 1929; Goldstein and Murphy, 1929). Intrau- 
terine exposure of the fetus to atomic radiation in sufficient amount to cause 
acute radiation eflfects on the mother has resulted in children with signifi- 
cantly smaller head circumferences than in the control group (Plummer, 
1952; Yamazaki ct al., 1954). Information concerning irradiation of the 
head alone in infants and children is scanty. Children irradiated for scalp 
lesions developed epilation in three weeks, and, after a year or so, hemi- 
paresis and siezures developed in one patient (Lorey and Schaltenbrand, 
1932) and a subdural hematoma was reported. In contrast to this, over 
3,000 persons, many of whom were children, recei\ing epilating doses of ra- 
diation for treatment of tinea capitis had no evidence of injury to the brain 
(Mackee and Cipollaro, 1946) . In another report, children over 3 years who 
received radiation to the head for a similar condition did not develop any 
abnormal neurologic findings (Macleod, 1909). Children under 3 years were 
not radiated to avoid any possibility of radiation injury. Periods of somno- 
lence lasting from 4 to 14 days were noted in 30 out of 1,100 children epi- 
lated by radiation therapy for ringworm of the scalp (Druckmann, 1929). 
A similar type of reaction was noted in adult human volunteers who received 
approximately 150 r to the diencephalic area (Birkner and Trautmann, 
1953). Disturbances of sleep-wake patterns and changes in gonadal function 
also were noted. In this regard, radiosensitivity of the hypothalamus and 
brain stem has been demonstrated in recent studies by Clemente and Hoist 
(1954) and Arnold and his associates (1954a,b,c). A report on the sur- 
vivors of the atomic bomb explosions who exhibited no sign of burns. 



EFFECTS OF HEAD IRRADIATION IN NEWBORN RATS 69 

trauma, or oeneralized radiation illness, but who were assumed to have been 
radiated to the head alone, revealed that the most extensive involvement of 
the brain was in children ( Uchimura and Shiraki, 1952). 

The relatively abrupt change in the degree of radiosensitivity of the neo- 
natal rat brain occurs at a time when unique morphologic, functional, and 
biochemical changes are taking place, and this seems to present an interest- 
ing temporal correlation with the findings reported in this study (Waelsch, 
1955: Richter. 1957). 

The general growth rate of the neonatal rat brain is reflected by the five- 
fold increase in weight by the end of the 2nd postnatal week and this repre- 
sents almost 809r of the weight of the adult brain (Potter ct al., 1945; Folch- 
Pi. 1955). The cerebral cortex is gaining weight proportional to the growth 
of the brain as a whole, but the cerebellum is gaining nearly three times as 
much weight, and the brain stem is accumulating only one-half its birth 
weight during the same period ( Sugino, 1917). However, the vascularity of 
the brain undergoes little change during the first 5 days, but between the 5th 
and 10th day a definite increase in vascular richness occurs. After the 10th 
day the density of the capillary bed increases rapidly, and a concomitant 
richness of the capillary bed increases in oxidase content and mitochondria 
simultaneously i Campbell, 1939). An example of the neuronal difTerentia- 
tion taking place during the neonatal period is presented by the change in 
the packing density of the neurons in the cerebral cortex. This density de- 
creased rapidly between the 3rd and 4th day after birth and then more 
slowly, "no change taking place after the 17th day" ( Haddara, 1956). This 
would indicate an increase in cytoplasmic constituents along with the devel- 
opment of a more elaborate cortical dendritic system. 

It is during the first 2 week period postnatally that the electroencephalo- 
gram becomes more regular and assumes the characteristics seen in adult 
rats 1 Grain, 1952). The ability of the rat to withstand anoxia is greatest in 
the immediate postnatal period, and shortly after birth during the first 5 to 
6 days there is a loss of tolerance to anoxia (Fazekas ct al., 1941 i. 

Immature budding vessels may well be more differentially sensitive to nox- 
ious agents than fully developed ones. It has been shown that growing retinal 
vessels during the first postnatal week, but not the choroidal vessels, constrict 
when exposed to oxygen and may e\en be obliterated with prolonged expo- 
sure ' Ashton and Cook. 1954). .\s the vessel reaches maturity, which takes 
place at about the 8th postnatal day in the rat. it gradually loses its ability to 
constrict when exposed to oxygen. In this regard, it is felt that the oxygen ef- 
fect is an appropriate example since oxygen enhances ionizing reactions 
(Dowdy et al., 1950) . Moreo\er, the best protectors against x-rays, i.e.. cystea- 
mine and cysteine, sulfhydryl containing amino acids, are also the best protec- 
tors of mammals against oxygen poisoning (Bacq and .Alexander. 1955) . How- 



70 YAMAZAKI, BENNETT AND CLEMENTE 

ever, it remains to be demonstrated whether radiation actually affects ma- 
turing cerebral vessels in a like manner. The capillaries have been demon- 
strated to be the most radiosensiti\e of the blood vessels, and the degree of 
vascularity would seem to have a bearing on the pathologic picture. The 
degree of vascularity varies widely in the rat brain; for example, the globus 
pallidus has been demonstrated to be strikingly low in vascularity (Craigie, 
1945). The globus pallidus was one of the areas where necrosis was most 
frequently involved. 

These physiologic and morphologic changes in the rat brain during this 
initial postnatal 2 weeks when radioresistance is rapidly developing are also 
associated with dynamic biochemical transformation. Thus, oxygen ixptake 
increases rapidly as does the lactic acid production ( Greengard and Mc- 
Ilwain, 1955; Tyler and Van Harreveld, 1942). Marked increase in enzyme 
activities occur; there is a threefold increase in the respiratory enzymes suc- 
cinic dehydrogenase and cytochrome oxidase during the initial 2 weeks and 
a fivefold increase of adenosine triphosphase activity, believed to be involved 
in making energy available to the cell to accomplish its differential growth 
(Potter, ct al., 1945; Flexner, 1953). Increased cholinesterase and pseudo- 
cholinesterase activity is similarly observed i Elkes and Todrick, 1955). The 
water content decreases significantly and levels of proteins and phosphatides 
are beginning to approach adult levels. Howe\er, the cerebrosides which are 
importantly related to myelin formation accimiulate only 119^ of the adult 
weight by the 19th day (Folch-Pi, 1955). Deoxyribonucleic acid content 
which continues to accumulate during this period increases fourfold between 
the 2nd and 16th postnatal days, after which further increase is hardly no- 
ticeable (Mandel and Bieth, 1951 ). These examples amply demonstrate that 
the x-irradiation may well alter these manifold biochemical changes occur- 
ring in the rat biain shortly after birth. 

Summary 

Newborn rats ranging in age from 8 hours to 15 days received single doses 
of x-radiation to the head only. Doses of 125 r, 300 r, 500 r, and 1.000 r 
were administered. One group of animals was sacrificed at 48 to 72 hours 
after irradiation, and another group was autopsied at ages ranging from 14 
days to 15 months. 

The study demonstrated the development of a marked radioresistance in 
the brain by the 3rd postnatal week, compared to the easily damaged brain 
of the rat during the first postnatal week. The development of this relative 
radioresistance following the first week of life was abrupt. The radiosensitiv- 
ity was manifested by an increased mortality, retarded growth, retarded 
brain size, production of cataracts, and abnormal neurologic signs in the 



EFFECTS OF HEAD IRRADIATION IN NEWBORN RATS 71 

radiated animals. The neuropatholosic findings correlated closely with the 
behavioral disturbances. Prominent among the early microscopic findings 
was damage to capillaries. Later histopathologic findings included perivas- 
culitis, progressive thickening of the \essel walls and a narrowing of the 
vessel lumen, meningitis, inflammation of the choroid plexus, ventricular en- 
largement, delayed cerebral cortical and cerebellar de\elopment. focal ne- 
crosis, and gliosis. 

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mammalian central ner\ous system. Proc. Soc. Exptl. Biol. Med. 80, 15-19. 
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EFFECTS OF HEAD IRRADIATION IN NEWBORN RATS 73 

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448-468. 



Cytoplasmic Inclusions Containing 

Deoxyribonucleic Acid in the Neural Tube 

of Chick Embryos Exposed to Ionizing 

Radiation"^ 

Mary Elmore Saier and Donald Dincax 

University of Texas Medical Branch. 
\ Galveston, Texas 

Introduction 

The identification of deoxyribonucleic acid ' DNA i with chromatin nor- 
mally confines it to the nucleus of the cell. Nevertheless, reports of the 
occurrence ot DXA in the cytoplasm are sufficiently numerous to suggest 
that its presence there may be a phenomenon of wide distribution, even 
thous:h the origin and destiny of such DNA may remain obscure. 

Perhaps not sufficiently appreciated is the large amount of cytoplasmic 
DNA which makes its appearance in young embryos a few hours tollowing 
moderate irradiation. The striking picture presented by the numerous Feul- 
^en positi\"e bodies in the neural tube of early chick embryos that had 
received moderate doses of ionizing radiation prompted the present investi- 
gation. This enlarges on a preliminary report t Sauer. 1957) dealing with 
irradiated chick embryos. Since similar bodies ha\e a normal occurrence 
Gliicksman. 1951: \on Sallmann ct al., 1957). their interpretation as an 
injury response demands special caution. In the chick, the large number and 
wide distribution ot these in irradiated embryos tar exceed any normal 
appearance. 

Materials and Methods 

Chick embryos ranged from 2 to 4/2 days in incubation age at the time 
of treatment, most being about 60 hours old. 

* Investigation supported in part by the U.S. Public Health Service. The authors 
gratefully acknowledge the cooperation of Dr. \Iartin Schneider, who supervised the 
.\-irradiation. and of Dr. Bruce E. Walker, who carried out the technique with the 
tritium-labeled enibrvos. C. Drew Sanders gave technical assistance. 



76 MARY ELMORE SAUER AND DONALD DUNCAN 

Embryos received either 200 or 500 r of x-rays through the unopened 
shell and were returned to the incubator. Control embryos were subjected 
to all treatment given the others, except the actual irradiation. Individual 
embryos were fixed at frequent intervals following irradiation, ranging from 
1 to 76 hours. The following factors were employed in the irradiation: 250 
kv, 30 ma, added filter 0.25 mm Cu plus 1.0 mm Al. 50 cm FOD, average 
intensity of 145 r/min. 

Other embryos were labeled with tritium ( Sauer and Walker, 1961). 
Some received only thymidine-H'*, usually 50 mc per embryo, specific activ- 
ity 1.6 c/mmole. Others had received 200 r x-rays immediately preceding 
treatment with thymidine-H'\ 

The embryos were fi.xed in Newcomer's (1953), Carnoy's. or Serra's 
fixative, or in 10 or 50 Cf neutral formalin, embedded in paraffin and sec- 
tioned at 3 to 6 IX. Stains used included hematoxylin and eosin, toluidine 
blue O, May-Griinwald and Giemsa, Feulgen's, and methyl green-pyronin. 
The May-Griinwald and Giemsa stain following Newcomer fixation was 
preferred for general cytologic study. Kurnick's ( 1955a,b) procedure was 
followed for DNase and RNase. The enzymes were purchased from Worth- 
ington Biochemical Corporation, Freehold, New Jersey. For electron micro- 
scopy, pieces of embryos were fixed in Dalton's osmic-dichromate mixture, 
embedded in methacrylate, and sectioned with a Porter-Blum microtome. 

Results 

Numerous cells in the irradiated embryos have a striking appearance in 
that one or more bodies approximately 2-5 /x in diameter lie within their 
cytoplasm (Figs. 2-12). Such bodies occur in small numbers in normal chick 
embryos but greatly increase in number following irradiation. In 2- to 3-day- 
old chick embryos, they may begin to appear in the 2nd hour after exposure. 
In the early hours after treatment the cells containing the inclusions may be 
few or many, depending on the susceptibility of the particular embryo. The 
distribution of the afTected cells at this time is wide but irregular: while 
some fields show many of the bodies, extensive regions may contain none or 
few. The bodies appear most constantly in the neural tube, especially in the 
brain. Their number increases with time, so that a 60 hour embryo that has 
received 200 r will 9 hours later contain large numbers of the inclusions in 
every system (Figs. 3-6). The body lies within the cytoplasm of an appar- 
ently normal cell. Its position adjacent to the nucleus is characteristic (Fig. 
6) and it often flattens or indents the nucleus at the area of contact. 

In the series receiving 200 r, the inclusions are \ery numerous in embryos 
fixed at 9 to 22 hours following irradiation. The number of inclusions then 
decreases rapidly. Only small numbers of the cytoplasmic bodies remain at 



CYTOPLASMIC DNA IN IRRADIATED NEURAL TUBE 77 

the end of 34 hours in any of the 200 r series, and by 57 hours recovery 
seems to be complete. Ahhough a variable amount of degeneration occurs 
even at this lower dosage, manifested in the neural tube as scattered nuclear 
fragments or as areas with an indistinct luminal margin, there is no whole- 
sale degeneration, and most nuclei remain essentially nonnal in appearance 
(Figs. 2-8). To what extent recovery consists of reversal of the process in 
the individual cell rather than replacement by unaffected cells can not be 
answered by this study. 

Quite different results follow a dosage of 500 r. At 20 hours after exposure, 
numerous inclusions fill many cells, and there is considerable cell death. 
Debris is present in the \entricles of the brain and in the central canal of 
the neural tube. This series shows considerable reduction in the mitotic rate 
during the 20 to 30 hour period following treatment, while no such sec- 
ondaiy delayed period of mitotic arrest follows the 200 r dosage. The 
series receiving 500 r shows complete recoxery at 76 hours. 

From the small series of older embryos i4 to 5 days i receiving the same 
amount of radiation as the two groups just described, it was determined 
that age is not a factor in the mere appearance of inclusions, although their 
distribution and time of maximum development differ in the two stages. 

Table I summarizes the staining reactions of most of the cytoplasmic 
inclusions. Each body commonly displays a deep staining region, or center. 
(Figs. 2 and 10) which is strongly basophilic and appears to contain a high 
concentration of DNA. Both with Feulgen's stain and methyl green-pyronin, 
the centers give a strongly positive reaction for DNA ; the negative reaction 
when DNase precedes the staining confirms the DN.A. content of the 
centers. 

Alfert I 1955; Vendrely ct al., 1958 i showed that reaction to methyl green 
is not a reliable indicator of the degree of polymerization of DNA, as held 
by Kurnick 1955a.b!. The intimate imion of DNA and protein in normal 
chromatin keeps many groups imaxailable tor dye binding. Although the 
DN.\ of pyknotic nuclei does not decrease until the fragmentation stage, 
the intense yreen which pyknotic nuclei display can not reflect the amount 
of DNA present, but only means that autolytic changes accompanying pyk- 
nosis have unmasked stainable groups. 

The staining reactions carried out ( Table I ) support the concept that 
these bodies also contain ribonucleic acid (RNA ) . With Kurnick"s 1955a, b) 
modification of the methyl green-pyronin stain, for which he claims speci- 
ficity for each of the types of nucleic acid, the body stains with a green 
center surrounded by a red periphery. The color resembles the deep green 
of the metaphases rather than the lighter green of other nuclei. The red color 
of the periphery might mean either RN.A or depolymerized DNA. The 
negative Feulgen stain and the absence of red color when the stain is applied 



78 



MARY ELMORE SAUER AND DONALD DUNCAN 

TABLE I 



Staining Reactions of the Bodies Which Appear in 
THE Cytoplasm After X-Irradiation 



Method 



Ceiitei 



Periphery 



Color 



Significance 



Color 



Significance 



Hematoxylin-eosin 

Toliiidine blue 

Feulgen's 
Omitting 

hydrolysis 
After DNase 
After RNase 

Methyl green- 
pyronin 
After DNase 

After RNase 



May-Griinwald- 

Giemsa 

After DNase 

After RNase 



Purple Basophilic 

Deep blue Presimiably 

nucleic acid 
Deep red 



No red 
No red 
Deep red 

Deep green 

No green 
on slide 
Deep green 



DNA 



DNA (polymerized? 



Black 

Black 

Mostly red, 
but a few 
remain black 



DNA and RNA? 



Red Acidophilic 

Pale blue — 

Green 

Green ) Not DNA 
Green 

Red 



Red 


) RNA (not de 




1 polymerized 


No red 


! DNA, for 




Feulgen- 




negative) 


Black or 


— 


gray 




Gray 


— 



after digestion with RNase indicate that it is RNA. The deep purplish black 
staining of the center or often of the entire body with the May-Griinwald 
and Giemsa stain would be consistent with this conclusion. Jacobson and 
Webb ( 1952) found that chromatin changes in its reaction to this stain dur- 
ing the sequence of mitosis from red in the interphase to black during meta- 
phase. These authors apparently demonstrate that black indicates presence 
of RNA in addition to DNA, but the specificity of this has been questioned 
(Swift, 1953; Theorell, 1955). In our hands black of the metaphase was 
more resistant to each type of nuclease than were the other cellular elements 
and that of the inclusions was even more resistant than were the metaphases. 
Following DNase, the bodies stained as black as before; following RNase, 
digestion was incomplete, some resistant bodies always remaining. 

The response of neural tube cells of embryos exposed for as long as 12 
hours to tritiated thymidine resembled the response to x-ray (Sauer and 
Walker, 1961). However, there was no cessation of mitosis in the tritium- 



CYTOPLASMIC DNA IN IRRADIATED NEURAL TUBE 79 

tieated embryos, wiiile mitosis was inhibited for IV2 and 3 hours fohowing 
the 200 and 500 r exposures respectively. Tritium, once added to the es:g, 
remains axailable over a prolonged period, so that a high degree of incor- 
poration results (Sauer and Walker, 1959. 1961 i. Assuming that the mitotic 
stages are the ones most sensitive to radiation, x-ray afTects only those in 
mitosis at the time of treatment. In the tritium-labeled embryos, howe\er, 
within the course of a few hours practically e\ery neural tube cell would 
undergo mitosis and thus be exposed to radiation at its sensitive stage. Those 
embryos exposed to the combined action of tritiated thymidine and 200 r did 
not appear to show much greater injury than those that had received thymi- 
dine alone. 

With only light microscopy, there remained the possibility of error as to 
the actual cytoplasmic location of the bodies. The electron microscope 
pro\ed in\aluable in demonstration of electron dense bodies of complex 
form not found in normal material 'Fig. 12 j. 

Discussion ^ 

Feulgen-staining granules located outside the nucleus ha\e been described 
in many species under a wide variety of conditions. They occur normally, 
especially in embryonic development 1 Gliickmann, 1951; Chang, 1940'. 
but also in the adult in certain locations (Corner. 1932) : pathologically fol- 
lowing cell death (Barthels and Voit, 1931) and in association with \iruses 
(Leuchtenberger et al., 1956) : and experimentally in tissue cultmes of nor- 
mal vertebrate embryos (Maximow. 1925) and in iriadiated material ' .'M- 
berti and Politzer. 1924: \on Sallmann ct al., 1957 . Wherever encountered, 
they resemble in their deep staining the chromatin of mitotic stages and are 
surrounded by a portion of cytoplasm more deep staining than the 
remainder. 

E.xtranuclear chromatin bodies assume prominence in developmental 
stages in both plants and animals in connection with death of superfluous 
cells, as in regression of transient structures or following excessive cell pro- 
duction. The latter is probably an almost uni\ersal growth phenomenon. 

In the intensely studied field of insect dexelopment. Feulgen-positive 
bodies regularly occur both within the cytoplasm and e.xtracellularly. Wig- 
glesworth (1942) in an extensive review established two facts: a) whole 
nuclei break down, and b ) the granules arc often intracellular. They are 
most numerous during active mitosis, when excess cells would be formed. In- 
corporation of the remnants of a dead cell by a neighboring cell seemed a 
distinct possibility in epidermis with its intercellular connections: also, in 
rapid cell division, of one of the daughter nuclei died before the cytoplasm had 
divided, the dead cell would remain as a cytoplasmic inclusion. In one 



80 MARY ELMORE SAUER AND DONALD DUNCAN 

known situation, nuclear division regularly occurs without cytoplasmic divi- 
sion, and one of the daughter nuclei degenerates to become a DNA-contain- 
ing cytoplasmic inclusion. Since origin from degenerating nuclei in this case 
is undisputed, Wigglesworth inferred such origin for all cases. Linder's 
(1956; Linder and Anderson, 1956) more recent observations support this 
conclusion. 

Gliicksmann (1951) in his classic review listed numerous descriptions of 
prominent, dark-staining bodies usually interpreted as degenerating cells in 
normal embryos. The Feulgen stain, whenever carried out, indicated nuclear 
material. Gliicksmann concluded that the bodies in all cases represented nu- 
clear degeneration which typically began with pyknosis, further changes 
occurring either in the isolated remnant or inside a neighboring cell that 
had resorbed it. Chang (1940) pointed out the large number and wide dis- 
tribution of the bodies in mouse embryos. He held that the bodies are within 
the cytoplasm, being phagocytized fragments of dead cells. According to 
Hamburger and Levi-Montalcini (1949), the entire body resembles a macro- 
phage in its reaction to vital stains. 

Nucleic acid normally moves from nucleus to cytoplasm by submicroscopic 
particles, but the same function may occasionally be accomplished by trans- 
port of large bodies. Here probably belong the examples of cytoplasmic DNA 
granules in certain plants (Sparrow and Hammond, 1947; Chayen and Nor- 
ris, 1953). A number of species of nematodes and insects undergo a chro- 
matin diminution process in connection with the segregation of the germ 
cells from somatic cells, whereby the somatic cells regularly cast out into the 
cytoplasm what may be a large part of the chromosomes (Wilson, 1934; 
Painter, 1959). 

Extranuclear DNA in nonexperimental pathologic states is usually inter- 
preted as degenerating nuclear remnants (Barthels and Voit, 1931). Other 
possibilities, especially in malignant cells, are nuclear buds which become 
enclosed in the cytoplasm, explained as an adjustment of the nuclear-cyto- 
plasmic surface ratio, and a direct extrusion of chromatin into the cytoplasm, 
leaving a hypochromatic nucleus (Ludford, 1942). Von Sallmann et al. 
(1955) in studying radiation-induced changes in the lens of laboratory ani- 
mals, where extranuclear Feulgen-positive bodies apparently are the pre- 
dominant pathologic finding, pointed out the striking analogy with age- 
induced changes. They considered the bodies to be extruded from the nu- 
cleus. Loewenthal (1957) interpreted the Feulgen-positive bodies found in 
large numbers in chick embryos homozygous for the "creeper" mutation as 
degenerating nuclei. Cytoplasmic inclusions containing DNA characterize a 
number of virus diseases. Leuchtenberger et al. (1956) recently applied 
electron microscopy and quantitative measurements of the DNA to the 
bodies constantly present in rectal polypoid tumors and concluded that they 
were viral. 



CYTOPLASMIC DNA IN IRRADIATED xNEURAL TUBE 81 

Maximow (1925) recognized that the peculiar granules occurring in tissue 
cultures of young rabbit embryos were inclusions within the cytoplasm of 
otherwise normal appearing cells, and that, except for their enormous in- 
crease in number, they were identical wdth the inclusions of normal em- 
bryos. They were especially abundant in the neural tube and mesenchyme 
and were usually more numerous in the central part of an explant. He stated 
that they always first appeared as small granules in close proximity to the 
nucleus, and he assumed that the large granules resulted from the growth of 
small ones. 

Temperature changes ( Chevremont it ai, 1958 1 and chemical agents 
(Dustin. 1947; McLeish, 1954: Chevremont ct ai, 1958) may evoke DNA- 
containing cytoplasmic bodies. Explanations have varied. Dustin (1947) saw 
evidence that dividing cells respond to colchicine and a series of other 
mitotic poisons by nuclear pyknosis. followed by fragmentation and engulf- 
ing of the debris by the cytoplasm of surrounding cells. "Micronuclei" may 
result from chromosome breakage and be the source of DNA-containing 
bodies in the cytoplasm. Chromosome breakage may occur from intracellular 
metabolic disturbances resulting from changes in temperature or o.xygen ten- 
sion (Roller, 1954) and following exposure to chemicals (McLeish, 1954; 
Frederic it al. 1959). Chevremont vt ai, (1958; Baeckeland it ai. 
1957) found that fibroblasts cultivated in the presence of DNase contained 
numerous Feulgen-positive granules in their cytoplasm. This DNA, which 
may amount to 90''r of the normal diploid nuclear value, was newly syn- 
thesized, as demonstrated by labeling with tritiated thymidine (Chevremont 
('/ al., 1959). Since other nonphysiologic agents, including chilling to 20°C, 
gave similar results (Chevreinont irt al.. 1958), and in \iew of the evidence 
that cytoplasmic DNA may mean only that dead nuclear fragments have 
been phagocytized, the authors" interpretation that the bodies are altered 
mitochondria must be accepted with caution. 

Extranuclear DNA following irradiation may be the result of cell death by 
nuclear pyknosis, ''micronuclei" resulting from chromosome breakage, or a 
manifestation of other mechanisms. 

According to Spear and Gliicksmann i 1938) and Gliicksmann i 1951 ). cell 
death from radiation injury is by pyknosis, and the Feulgen-positive bodies 
represent pyknotic degeneration, which they subdivided into three stages: 
chromatopycnosis, consisting of the separation of the chromatic from the 
nonchromatic material and the precipitation and coalescence of the chro- 
matin into granules; hyperchromatosis of the nuclear membrane, in which 
the chromatin, having united into a single body, lies against the nuclear 
membrane as a deeply staining rim or partial rim; and chromatolysis, with 
loss of the Feulgen reaction. The entire process may take place in about an 
hour. Since onset of prophase in itself effects separation of chromatic from 
nonchromatic elements, cells degenerating in mitosis omit the first stage. 



82 



MARY ELMORE SAUER AND DONALD DUNCAN 



This behavior furnishes a means of distinguishing, on the basis of the Feul- 
gen-positive granules, between death of mitotic and of interphase stages. The 
granules become cytoplasmic when resorbed by a neighboring cell. 

In Fig. 1, based on the neural tube of the irradiated tadpole during the 
prolonged prophase extending from 11 to 24 hours, the low metaphase count 
means that heavy casualties occur on completing prophase. These casualties 



5 ^5 

V 

O 
o 50 

o 
S 25 



2 50 



« 25 



I 


Prophase 




Xi^ ^ V 

1 Metaphase "^ -— ~__^ 




'•■. \ , — 


^ 


•.\^.-' Telophase 




/ \ 


II 


/ B. Hyperchromatosis 
'A \ C. 'Lysis> 




/K 


/ ^^"^-^ '^ — ^^._. 


/;% 


',^^/ A. Pyknosis "^»^,^ 



8 



40 



48 



16 24 32 

Hours after irradiation 
MODIFIED FROM SPEAR & GLUCKSMANN, (1938) 

Fig. 1 . Chart showing the number of mitotic and degenerate cells in the brain and 
retina of young tadpoles for 48 hours after exposure to gamma radiation (268 r). 

\. Mitosis. Key: prophase, ; metaphase, ; anaphase and telophase, 

Although prophases are normally fewer than metaphases, from 11 to 24 hours after 
radiation the number of prophases greatly exceeds the number of metaphases, indi- 
cating a prolongation of the prophase period. The subsequent decrease in the number 
of prophases without change in the number of metaphases indicates degeneration of 
many of the prophases. II. Degeneration: Three stages, as applied to the nucleus. 
Stage A. Chromatopycnosis, . The chromatic material separates from the non- 
chromatic, with the chromatin material appearing as scattered granules. Stage B. 

Hyperchromatosis of the nuclear membrane, . The chromatin granules have 

united into a single, deeply staining mass sitting as a cap on the nuclear membrane. 

Stage C. Chromatolysis, The chromatin breaks into fragments, and loses its 

Feulgen staining properties. 

Since the mechanism of prophase effects separation of chromatic from non-chromatic 
material, a cell which reaches prophase before degenerating omits the first stage and 
passes directly into hyperchromatosis. The high increase in hyperchromatosis, be- 
ginning shortly after the peak of the prophase curve, indicates that the greatest num- 
ber of casualties occurs after the cell reaches prophase. (Modified from Spear and 
Gliicksmann) . 



CYTOPLASMIC DNA IN IRRADIATED NEURAL TUBE 83 

cause a rapid rise in the degenerate cell count, beginning at about 13 hours, 
and since the dying cells are mitotic stages, this rise is in the hyperchromatic 
type of granule. The smaller rise in the pyknotic stage shows that some cells 
also break down before beginning division. 

So-called micronuclei are a well known and classic effect of radiation. 
Although first seen by Koernicke ( 1905) in irradiated roots, it remained for 
Alberti and Politzer ( 1924) to show in the corneal epithelium of salamander 
lar\ae what becomes of a piece broken from a chromosome if it does not 
rejoin. Lacking a centromere, it does not move to one of the poles in ana- 
phase, but lags on the spindle and is usually not included in either daughter 
nucleus at telophase. Remaining in the cytoplasm, it becomes spherical and 
lies beside the chief nucleus. They named these Teilkerne or partial nuclei. 
Se\eral isolated chromosome fragments may unite into a single larger micro- 
nucleus ( Ohnuki and Makino, 1960). 

The great majoritv of so-called micronuclei are nonliving, spherical, deep 
stainins; bodies about 2 /x in diameter. Those few acentric fragments that 
contain both an adecjuatc amount of heterochromatin and a nucleolar or- 
ganizer may continue to li\e, however, playing an active part in cell metabo- 
lism and dividing synchronously with the main nucleus for several subse- 
cjuent life cycles i LaC^our, 1953: McLeish, 1954'). The completeness of the 
chromosome set is supposedly necessary for the normal functioning of the 
cell; consequently, the daughter cell with the deficient chromatin, presum- 
ablv the one in whose cytoplasm the micronucleus became enclosed, has been 
assumed to be short-lived. However, Ohnuki and Makino (1960) pro\ed 
sur\i\al throughout at least one mitotic cycle, and Hornsey 1956, 1960) 
showed that their ma.ximum number appeared at the end of the first 
mitotic cycle ( prolonged by irradiation ) and that their subsequent decrease 
was exponential, depending only on dilution by further cell di\ ision. 

Chromosome breaks may occur trom exposure ot a cell to irradiation in 
any stage of its life cycle. However, interphases in which the chromosomes 
are widely dispersed show a special resistance to chromosome breakage as 
compared to cells irradiated in the premitotic and mitotic stages when the 
chromosomes are tightly condensed. Muller (1954) reviews the factors in- 
\ol\ed. Lagging chromosome fragments are the chief change resulting from 
irradiation in interphase, being few in early stages and becoming more abun- 
dant as interphase progresses. Chromosomes irradiated in late prophase to 
early telophase, even though eflfectively broken, give no e\idence of being 
broken at the time, for when in the condensed condition they are held as if 
by some enveloping material and can not fall apart into fragments. How- 
ever, when the chromosomes recondense at the next mitosis, after an inter- 
xenina: interphase has elapsed, more structural changes appear than would 
ha\e followed irradiation in the interphase. Consequently, micronuclei are 



84 



MARY ELMORE SAUER AND DONALD DUNCAN 








3 



^ 



'^ms 



CYTOPLASMIC DNA IN IRRADIATED NEURAL TUBE 85 

rare until cells ha\e undergone at least one mitosis subsequent to irradiation. 
Micronuclei are more prominent in some material than in others (La Cour, 
1953). Both the number of fragments per cell and the number of cells with 
fragments increase with the dose ( Roller. 1947) . At their height, micronuclei 
may occur in lO^r or more of cells (Gray and Scholes, 1951 : von Sallmann, 
et al, 1957; Friedkin, 1959). 

Most ideas in the literature as to the nature of the bodies in the cytoplasm 
are based on interpretations of a static picture rather than on direct obser- 
vation. Of indisputable origin are micronuclei. Recordings with time-lapse 
photography have been made of the formation of micronuclei in irradiated 
cells and of their movement into the cytoplasm (Bajer, 1958; Bloom ct al., 
1955: Ohnuki and Makino, 1960). The origin of a chromatin body in the 
cytoplasm through degeneration of a sister nucleus in a cell which did not 
complete division is well founded in a restricted field ( VVigglesworth, 1942) 
and has also been observed in irradiated tissue cultures (Stroud and Brues, 
1954). Direct extrusion of nuclear material into the cytoplasm has ofen been 
postulated as the method of formation of these bodies, but apparently has 
not often been observed in irradiated material, nor has the gradual growth 
of a cytoplasmic body de novo in the cytoplasm, nor actual phagocytosis of 
degenerated nuclei. 

Our material confirms the observations of others (Butler. 1936; Schneller, 
1951) working with irradiated chick embryos. Attention has been centered 
on the large amount of extranuclear Feulgen-positive material present, espe- 
cially in the neural tube. Apparently the presence of DNA in the cytoplasm 
is not a universal reaction to irradiation. Mitchell !l942) found the cyto- 
plasm of irradiated tumor cells consistenly negative to the Feulgen reaction. 

Our material justifies the conclusion that many of the bodies lie within 
the cytoplasm (Figs. 2-1 1 ) . This could often be demonstrated with the light 
microscope with Zeiss stereoscopic eye caps to give an exaggerated view of 
depth. The electron microscope pictures are indisputable (Fig. 12) Wanko 
etal.,\959. 

Spear and Gliicksmann's i^l938j and Glucksmann's (^1951) distinction be- 
tween two types of chromatin bodies, depending on whether death of the 
cell occurred in interphase or in mitosis, aids in identification of certain 

Fig. 2. Feulgen stain of the neural tube of a 3-day chick embryo which had re- 
ceived 200 r of x-rays 7 hours previously. Many of the cytoplasmic bodies are of com- 
pound nature (see arrows) containing one or several Feulgen-positive centers, accom- 
panied by Feulgen-negative material. Compare Fig. 10. Newcomer fixative. X 1200. 

Fig. 3. Brain of a 2'/2- to 3-day chick embryo which had received 200 r of x-rays 
9 hours previously. The lumen is at the top of the figure. This field shows only minor 
change compared to much of the embryo. Carnoy fixative : May-Griinwald and Giemsa 
stain. X 1000. 




Figs. 4-6. From the same brain as Fig. 3 (2/2- to 3-day chick embryo, 200 r, 9 
hours). Carnoy fixative; May-Griinwald and Giemsa stain. 

Fig. 4. The lumen is at the top of the figure. X 1100. 
Fig. 5. The lumen is at the top of the figure. X 800. 
Fig. 6. Enlargement of cell indicated in Fig. 5. X 3000. 



86 







Fig. 7. Brain of a 2yi>-day chick embryo whicti had received 1^00 r of x-rays 1 1 Va 
hours preceding fixation. Lumen is at the right. Newcomer fixative; May-Griinwald 
and Giemsa stain. X 900. 

Fig. 8. Neural tube of a 2'/;- to 3-day chick embr>'o which had received 200 r of 
x-rays H'/j hours previously. Newcomer fixative: May-Griinwald and Giemsa stain. 
X 1000. 

87 



88 MARY ELMORE SAUER AND DONALD DUNCAN 

bodies. Those which are dish-shaped, lens-shaped, broken ring-shaped, and 
possibly also those consisting of several granules, are features of the neuro- 
epithelial layer during the period of highest mitotic activity (8 to 15 hours 
after 200 r) when numerous cells break down in division. This period is 
approximately coextensive with the first mitotic period following irradiation. 
Since these bodies occur at all depths of the neuroepithelial layer, while 
dead cells are chiefly observed adjacent to the lumen, the bodies must have 
been moved into the depths of the wall following cell death. Although some 
are cast into the central canal, it may be speculated that others are resorbed 
at the lumen into the cytoplasm of adjacent nuclei which are beginning 
their postmitotic migration peripherally (Sauer, 1935; Sauer and Chitten- 
den, 1959; Sauer and Walker, 1959: Sidman et al, 1959; Watterson et al, 
1956). Evidence for the actual process of phagocytosis is completely lacking 
in sectioned material; however, to one who has marveled at the antics of 
cells in tissue cultures, as demonstrated with time-lapse photography, rapid 
absorption of degenerated cell remnants seems plausible. The many chro- 
matin bodies surrounded by only intact cells can be explained readily in 
this way. 

In addition to the irregular bodies which seem to be degenerated mitotic 
cells, after irradiation there are also larger, Feulgen-positive bodies more 
nearly approaching a cell nucleus in size. After 500 r these are the first 
bodies to appear in the neuroepithelial layer. They begin about the time 
of resumption of mitosis and become numerous 4 to 5 hours following irra- 
diation (Fig. 9) . The picture is clean-cut, in contrast to the same region seen 
overlain with small fragments at 10-14 hours following 200 r. Similar large, 
spherical bodies occur in enormous numbers in the mantle layer of our older 
irradiated chick embryos and of irradiated Chinese hamster embryos of com- 
parable age. Their large sizes suggests whole pyknotic nuclei. It is assumed 
that they are differentiating cells which have died a pyknotic type of death 
in interphase. Since they begin to appear even in the period of mitotic arrest, 
they are unrelated to mitosis. (Hicks, 1958 and Hicks et al., 1959) has studied 
this radiosensitive form extensively. Some in the neuroepithelial layer are 
definitely cytoplasmic. In the mantle layer, few intact cells remain. 

Those Feulgen-positive bodies definitely enclosed in mitotic stages at the 
lumen are considered to be micronuclei. In some material they are rather 
numerous. As the nuclei of these mitotic cells migrate into the depths of the 
wall in their postmitotic period, their micronuclei will be drawn with them. 
Consequently, some of the mitotic bodies deep in the wall must also be 
micronuclei. 

How many of the numerous, spherical bodies, about 2 /x in diameter and 
apparently cytoplasmic, are actually micronuclei is unknown. That some 
may represent a direct extrusion from the nucleus is a possibility. This might 



CYTOPLASMIC DNA IN IRRADIATED NEURAL TUBE 



89 






9-. ■• 



11 




90 



MARY ELMORE SAUER AND DONALD DUNCAN 










Fig. 12. Electron microscope picture of a tangential section of the neural tube. 
X 15.000. Line indicates 1 micron. 

not represent e.ssential DNA but an excess formed in a period of derant^ed 
metabolism or the reversal of a synthesizinc; cell to its presynthetic stage. A 
related idea is an accumulation in the cytoplasm of nuclear material which 
difTuses freely but is normally present in only small amount. RNA increases 
in the cytoplasm followin" irradiation (Mitchell, 1942). Consequently, more 
DNA from the nucleus is transforming into RNA of the cytoplasm. Some of 
the cytoplasmic bodies might result from a derangement in this process. 

This has been a fascinating study, but it has raised more questions than 
it has answered. What is the potentiality of early neural tube cells for resoip- 



FiGs. 9-11 are on page 89. 

Fig. 9. Feulgcn stain of the brain of a 4J/j-day chick embryo iriadiated with 500 r 
3 hours previously. The lumen is at the top of the figure. Newcomer fixative. X 750. 

Fig. 10. Feulgen stain of the brain of a 3- to 3'/; -day chick embryo which had 
been irradiated with 500 r 3J/2 hours pre\iously. The lumen is at the top of the figure. 
Most of the bodies contain both Feulgen-positive and negati\e regions. Carnoy fixative. 
X 1450. 

Fig. 11. Neural tube of a 2J/2-day chick embryo irradiated 8 hours previously with 
500 r. The lumen is at the left. Newcomer fi.xative; May-Griinwald and Giemsa stain. 
X 1400. 



CYTOPLASMIC DNA IN IRRADIATED NEURAL TUBE 91 

tion of contieuons solid material? What use is made of all of this cytoplasmic 
DNA? Surely a ready made supply of its own brand of DNA is a material 
too valuable to rapidly di\idino cells to be wasted. What is the role of 
DNase, or its absence, in connection with cytoplasmic DNA? Finally, there 
remains the unsolved problem of the great radiosensitivity of the differentiat- 
ing neinoblast. What change has suddenly come over this cell, probably still 
with mitotic potentiality, to increase its sensitivity and, with neurofibril ap- 
pearance, to lea\e it again to make it among the most resistant of cells? 

Summary 

The material consisted of 2- to 4-day chick embryos subjected to 200 to 500 
r of x-irradiation or labeled with thymidine-H"* of high specific acti\ity. 

Moderate doses of radiation led to a structural change in the cytoplasm 
in numerous cells of the early neiual tube. A striking featvne a few hoins 
following exposure to ionizing radiation was the presence within the cyto- 
plasm of one or more dense, basophilic bodies approximately 2-5 fx in diam- 
eter. These typically consisted of one or several Feulgen-positive centers, 
surrounded by an RNA-containing rim. The centers were digested by DNase. 
They represent a relatively large amount of extranuclear DNA. Electron 
microscopy demonstrated their great density and confirmed their cyto- 
plasmic location. At the lower dosage of x-ray, the process was completely 
reversible, without an inteivening period of degeneration. The bodies were 
not confined to the neural tube, although they attained great prominence 
there, but were widely distributed throughout the embryo. 

It is concluded that the bodies are of se\eral types. It is possible to dis- 
tinguish between those resulting from degeneration of mitotic stages and 
those of interphases on the basis of their morphology. Micronuclei are an- 
other type, sometimes present in considerable number. 



Refp:rences 

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Alfert, M. 1955. Changes in the staining capacity of nuclear components during cell 
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McLeish, J. 1954. The consequences of localized chromosome breakage. Heredity 8, 

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Carnegie Inst. Wash. Publ. 16 (80). 47-110. 
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Sauer, M. E., and Chittenden, A. C. 1959. Deoxyribonucleic acid content of cell 



94 MARY ELMORE SAUER AND DONALD DUNCAN 

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Macmillan, New York. 



Biochemical Effects of Irradiation in the 
Brain of the Neonatal Rat* 

O. A. ScHjEiDE. J. N. Yamazaki, C. D. Clemente. Nancy Ragan, and 

Sue Simons 

University of California School of Medicine, Los Angeles; 
and V.A. Hospitals, Los Angeles and Sepidveda. California. 

Introduction 

From several perspccti\es the brain of the neonatal rat may be regarded 
as embryonic. The cortex and the cerebellum are relatively small compared 
to the brain stem (the more basic and primitive structure), and even in the 
brain stem the nerve tracts are incompletely myelinated ( Folch-Pi, 1955). 
The cell nuclei are lartjer and more hydrated than adult nuclei, and the 
neurons have not yet assumed their characteristic elongate and dendrite 
configurations. During the first 2 weeks, extensive cell division takes place in 
the cerebellum and cortex. In the brain stem cells increase greatly in total 
volume, but there is less cell division. 

Hicks I 1952 I has shown that neuroblasts in the brains of newborn rats 
may be exposed to more than an hour of total anoxia without breakdown, 
a finding that indicates a well developed glycolytic mechanism and relates 
these structures metabolically to other embnonic tissues (Gal rt al., 1952). 

Aside from purely practical applications of studies on irradiation damage 
to brain tissue in young animals, these embryonic properties of the brains 
of neonatal rats make them useful tools in the assay of radiation effects on 
cell mechanisms in general (and on ner\ous tissue in particular). The neo- 
natal brain presents a continuously changing aspect over a period of weeks. 
During this time radiation-produced lesions may manifest themselves in 
various ways. C'.onversely. certain mechanisms of difTerentiation may be 
further elucidated by the changes induced by radiations given soon after 
birth. 

The present work is a preliminary sur\ey of the effects of sublethal doses 
of x-irradiation to the heads of newborn rats. Several aspects of brain mor- 
phology, biochemistry, and metabolism have been followed up to 40 days 
after birth. 

* These studies were partially supported l)y the .\tomic Energy Commission. 

95 



96 SCHJEIDE, YAMAZAKI, CLEMENTE, RAGAN AND SIMONS 

Materials and Methods 

Litters of Wistar strain rats were di\ided into two "roups. The individuals 
of one group received 750 r of x-irradiation to the head only (the pituitary 
was irradiated as well as the brain ) at 2 days of age and were kept for vary- 
ing lengths of time before sacrifice. Individuals of the other group served as 
litter-mate controls. 

At sacrifice (by decapitation), the brains of all rats were separated into 
three precisely resolvable parts: brain stem, cerebellum, and cortex. Each 
sample was frozen and stored in a freezer. On thawing, the total wet weights 
of the organs were determined, as were total water contents, total solids, 
total nitrogens, total lipids, and total phospholipids. Selected samples were 
analyzed for wet volumes of nuclei, mitochondria, and microsomal fractions; 
total lipids were extracted and their fatty acid profiles obtained by gas 
chromatography. 

Water content was determined by disintegrating the thawed tissue in a 
piston-type disintegrator, adding acetone to a weighed sample of the wet 
mash, evaporating thrice under a stream of nitrogen at room temperature 
(Sperry, 1955), and then drying in an Abderhalden apparatus over boiling 
water for 3 hours in the presence of PO5. Water loss was measured gravi- 
metrically. Total lipid was extracted from the dried solids by addition of 
10 ml of a 2:1 chloroform-methanol mixture. The extract was pvnified by 
Sperry's modification of the procedure of Folch-Pi rf al. ( Spen7, 1955), the 
total lipid being measured gravimetrically. Total phospholipids were deter- 
mined gravimetrically by extracting the total lipids with 15.0 ml of acetone 
to which one drop of saturated MgClj had been added. Nitrogen analyses 
on the solid residue remaining after lipid extraction was done by the micro- 
Kjeldahl technique. 

Subcellular components were isolated by differential centrifugation, and 
measurements of wet volumes of the fractions were carried out as detailed in 
a previous publication ( Schjeide, d al., 1960). 

Gas chromatography of the fatty acids derived from nuclei and mito- 
chondria^ was performed on the Model 10 Barber-Coleman apparatus, using 
a 50-in. column packed with commercially obtained ethylene glycol suc- 
cinate at 170°C. The detector was of the argon-ionization type. A 3 /aI 
aliquot of 2.5% in petroleum ether solution was injected into the column 
for each analysis. 

' The methyl esters of these fatty acids were prepared by solubilizing the total lipid 
in 1 ml of dried benzene, placing this in a 15-ml glass-stoppered centrifuge tube to 
which was added 2 ml of 4% anhydrous methanolic HCl, refluxing 4 hours over 
methanol, extracting the pentane-soluble lipids in 10 ml of pentane, washing thrice 
with distilled H2O, shaking with magnesium sulfate (to remove residual H:;0), and 
finally absorbing away the nonesterified lipids on powdered alumina. 



BIOCHEMICAL CHANGES IN NEONATAL BRAIN 



97 



Results 

As the brains of youno or irradiated rats were removed from their brain 
cases, they appeared less firm and more hydrated than the brains of old or 
nonirradiated rats. 

Some of these tissues were analyzed immediately after removal from the 
animal. The brains of the youngest rats ( 7 days ) contained an average of 
88.0^/h water. No modifying effect of x-irradiation was noted. By the time 
the rats were 40 days old, the cerebellums and cerebral cortexes contained 
80.0*^0 water and the brain stems only TS.O^r. X-irradiation elevated the 
water content to SO.Orf in the brain stems. Only slight hydration was noted 
in irradiated cerebellum and cortex. 

Figures 1, 2, and 3 illustrate the efTects of radiation on the total dry 
weights of brain stem, cortex, and cerebellum. 

During the first 2 weeks after 750 r of x-irradiation, enlargement of the 
brain stem followed a normal course, but then the growth rate became some- 
what depressed in all groups of animals ( Fig. 1 ) . The dry weights of irradi- 
ated cerebral cortex were comparable to those of control rats until 16 to 20 



90 
80 



Mgs. 




• Control 
O X-Ray 



t 
X-Ray 



15 
Days of Age 



30 



Fig. 1. Dry weights of brain stems from control and x-irradiated neonatal rats. 

days after exposure. At this time they fell off markedly (Fig. 2) . Cerebellums 
were profoimdly affected by radiations, retardation in growth being evident 



98 



SCHJEIDE, YAMAZAKI, CLEMENTE, RAGAN AND SIMONS 























4 


170 


• 
















/ 


/ 


160 


■ 
















/ 

• 


r 


150 


■ 




















140 


• 




















130 


■ 








o 


• 


/ 








120 


• 








• 




/ 




O 


< 


110 
Mgs. 










• / 


o 










100 










/ 












90 


• 






• 


/ 












80 


• 




















70 


• 








o 










< 


60 


• 






X* 














50 






/ 
















40 




/ 


/ 
















30 




/ 










• 
o 


control 
X-Ray 






20 























10 










1 


1 











X-Ray 



15 
Days of Age 



20 



Fig. 2. Dry weights of cortexes from control and x-irradiated neonatal rats. 



during the first week after x-irradiation (Fig. 3). 

Figures 4 and 5 show that the amounts of total Hpid per unit dry weight 
of cortex and cerebeUum increased at the same rates with increasing age 
despite irradiation. Obviously, both total lipid and phospholipid were re- 
duced per whole brain in those animals that displayed decreases in brain 



BIOCHEMICAL CHANGES IN NEONATAL BRAIN 



99 



Mgs 




15 
Days of Age 



Fig. 3. Dry weights of cerebellums from control and x-irradiated neonatal rats. 



dry weights following radiation exposure. Preliminary analyses for total 
phospholipid revealed no consistent differences either among the different 
brain com{X)nents or between control and irradiated animals (phospholipid 
averaged approximately 80^r of the total brain lipid in both cases). How- 
ever, the unit lipid content of irradiated brain stern was decreased signifi- 
cantly at 16-20 days of age (Fig. 6). This inhibition of lipid synthesis was 
closely proportional to a retardation of normally occurring dehydration of 
the whole brain stem tissue. 



100 



SCHJEIDE, YAMAZAKI, CLEMENTE, RAGAN AND SIMONS 



O • • 
• 40 O 



• O 



O • 
O • 



< 

O 

09 

in >, 






BIOCHEMICAL CHANGES IN NEONATAL BRAIN 



101 



o • o* 

go 9b 






o 3 



O GO 

•8 



o • 



102 



SCHJEIDE, YAMAZAKI, CLEMENTE, RAGAN AND SIMONS 




• O 



Nitrogen values for lipid-extracted brain solids were obtained for rats 
aged 11-18 days. In nineteen groups, no significant differences could be 
demonstrated either as a function of age or of x-irradiation. There was an 
average of 83% protein and amino acid in the lipid-extracted solid material. 
The potentially most interesting stages with respect to variations in intra- 



BIOCHEMICAL CHANGES IN NEONATAL BRAIN 



103 



cellular nitrogen (and DNA), namely rats less than 1 week of age, have not 
yet been studied. 

Proportions of centrifugally isolated nuclei, mitochondria, and microsomes 
for cortices and brain stems of various age groups (with and without prior 
x-irradiation) are presented in Table I. Mitochondria in the cortex ap- 
peared to increase per unit cellular volume with increasing age (and devel- 
opment). The only consistent changes following irradiation appeared to be 
decreases in wet volumes of mitochondria in some of the younger (8- to 
9-day) and older (28- to 30 day rats). However, since electron microscope 
examination of brain mitochondrial fractions obtained by the technique of 
Schneider and Hogeboom (1950) reveal the presence of materials other 
than mitochondria, the present results can only be regarded as tentative. 

Gas chromatograms of fatty acids from nuclei and mitochondria in 



TABLE I 

Wet V'olu.mes of Intracellvlar Components of Cortex and 
Brain Stem, with and withott Irradiation 



Sumber 
















of 


Age 
(days) 


Organ 


Treatment 
(in r) 


V 


olurnes (Ml/4 ; 


ml)" 




animals 


Nuclei 


Mitochondria 


I Microsomes 


6 


8 


cortex 


control 


0.20 


1.24 




0.92 


9 


8 


cortex 


750 


0.21 


1.04 




0.80 


5 


8 


cortex 


control 


0.48 


2.84 




1.60 


5 


8 


cortex 


750 


0.30 


1.80 




2.00 


5 


8 


brain stem 


control 


1.20 


4.80 




.56 


5 


8 


brain stem 


750 


0.40 


2.24 




2.64 


11 


9 


cortex 


control 


0.39 


1.07 




1.00 


12 


9 


cortex 


750 


0.34 


1 .00 




.80 


5 


16 


cortex 


control 


0.76 


2.80 




1.96 


4 


16 


cortex 


750 


0.80 


2.96 




1.80 


4 


20 


cortex 


control 


0.30 


2.42 




1.60 


5 


20 


cortex 


750 


0.42 


2.40 




1.90 


4 


20 


brain stem 


control 


0.75 


2.66 




1.50 


3 


20 


brain stem 


750 


0.40 


2.60 




0.68 


6 


28 


cortex 


control 


0.78 


4.80 




1.91 


4 


28 


cortex 


750 


1.36 


3.73 




1.60 


4 


30 


cortex 


control 


0.32 


4.05 




2.02 


3 


30 


cortex 


750 


0.27 


2.68 




3.46 




30 


cortex 


control 


0.30 


1.80 








30 


cortex 


750 


0.36 


1.44 







*MI per inl4 of starting tissue. In some ccises the sum of the wet volumes of the intracellular com- 
ponents exceeds that of the starting tissue. This phenomenon is due to uncontrolled hydration of the 
solids and inefficient packing in the volumeti ic tubes. 



104 SCHJEIDE, YAMAZAKI, CLEMENTE, RAGAN AND SIMONS 

cerebral cortices of rats aged 8-9 days to 30 days appear in Fig. 7. The 
most prominent fatty acids in these cross sections include palmitic, stearic, 
oleic, arachadonic, and linoleic, in that order. Experience with fatty acids 
from the developing livers of chicken embryos has shown that, as the cell 
matures, the percentage of palmitic acid in the nucleus decreases and the 
percentage of oleic acid increases markedly (Schjeide, 1960). X-irradiation 
retards the adjustment of these fatty acid ratios, and it may that this is a 
reflection of inhibition of differentiation in the nucleus. Re-enforcing this 
finding is the observation that the fatty acid profiles of mitochondria from 
irradiated chicken embryos contain an increased percentage of oleic acid. 

Although the nuclear fatty acids of neonatal rat brains failed to show 
changes as dramatic as those observed in the embryonic livers, there was 
generally a decrease in the ratio of palmitic acid to stearic acid and oleic 
acid in nuclei as a function of increasing age (Fig. 8). In cortices of all 
stages the decrease of this ratio was retarded by a dose of 750 r (Fig. 8). 
The decrjease in ratio was also retarded in nuclei of irradiated brain stems 
(Fig. 8). Significantly, control fatty acid ratios of the nuclei in brain stems 
(the more mature portion of the brain at this early age) were those assumed 
to be more characteristic of adult-type nuclei (Fig. 8). 

Linoleic acid in brain lipids decreased with age, reflecting the develop- 
ment of the "blood brain barrier." Irradiation did not appear to have any 
consistent effect on the percentage of linoleic acid in the brain lipids. 

Discussion 

Although the foregoing represents a purely introductory survey of bio- 
chemical effects of radiation on maturing brain, three points of interest 
stand out at this early stage. 

First, per unit dry weight of cortex and cerebellum, the total lipid, total 
phospholipid, and total nitrogen appear relatively unchanged by exposure 
of the heads of rats to 750 r of x-irradiation. Thus, in these respects, the irra- 
diated neonatal cortex and cerebellum can be considered essentially as 
miniatures of their control counterparts, difTering primarily in having fewer 
total cells per organ and having these cells poorly arranged in the greater 
structural context. A similar situation appears to exist in the deformed skele- 
tons of rat embryos irradiated between 12 and 16 days of gestation (Russell, 
1954). As far as is known, calcification mechanisms of the structurally-poor 
skeletons are unimpaired by the radiations that initiated the deformity; i.e., 
the enzymes involved in calcification appear to be present in sufficient quan- 
tity in irradiated bone. 

Contrasting with the lack of biochemical effects of radiations hereto ob- 
served on cerebellum and cerebral cortex is the inhibition of lipid synthesis 



BIOCHEMICAL CHANGES IN NEONATAL BRAIN 



105 




.jJk,^ 




Fig. 7(a) Fatty acid profiles of nuclei from cells of 8-day cortices. Solid line = 
control pattern. Dotted line=:pattern from x-irradiated cortices. 

Fig. 7(b). Fatty acid profiles of mitochondria from cells of 8-day cortices. Solid 
line=control pattern. Dotted line=pattern from x-irradiated cortex. 



106 



SCHJEIDE, YAMAZAKI, CLEMENTE, RAGAN AND SIMONS 



1:1.30 


■ 






A ^ Brain 

/ 


stem 




1:1.40 








/ 

A 






Ratio 














1:1.30 












•^. 


1:1.20 












^^ ,© 


1: 1.10 












.--'' 




• 


• 


.--?-t>- 


-'"' 


A« 


Control 


1:1.00 




o 


1:0.90 


©""" 
















A© 


X-Ray 


1.0.70 















16 17 20 
Days of Age 



Fig. 8. Ratios of palmitic acid to combinL-d total of stearic and oleic acids in nuclei 
of control and x-irradiated neonatal rat cortices and brain stems. 



in the brain stem. Because of the important relationship that has been (in- 
directly) established between the neural cells and closely adjacent oligoden- 
droglia cells with respect to the production of myelin (lipid), it would 
appear to be important to determine histologically the ratio of oligodendrog- 
lia to neural cells in irradiated brain stem, for in this organ complex, not 
only does the inhibition of lipid synthesis correlate closely with the retarda- 
tion of dehydration, but the inhibition is first noted (ca 16 days) at a point 
in development when a large increase in myelination processes takes place. 
These results point up the fact that the various sections of the brain are by 
no means homogeneous tissues and offer the possibility that in brain stems 
the oligodendroglia are vulnerable to radiations at a time when the strictly 
neural elements may be relatively less so. (Such vulnerability could be ex- 
pressed either as outright destruction of the cells or alteration of their syn- 
thetic abilities.) Histological studies currently being carried out by one of 
the authors of this paper (C D. Clemente) should help in the elucidation of 
this issue. 

A second point of interest concerns the radiation-induced decreases in 
mitochondrial populations as obserxed in the cortices of some younger and 



BIOCHEMICAL CHANGES IN NEONATAL BRAIN 107 

older rats. Such an eflfect is seen in cells of the embryonic chicken liver 
(Schjeide, 1960) and is thought to be indirect because of the time required 
(3 days) for the fall in population. It remains to be determined whether the 
decrease in mitochondria observed in the neonatal rat cortex is consistent 
and due to direct effects, to damage to a controlling mechanism in the 
nucleus, or to elicitation of toxic blood-borne factors. 

A third interesting result which may be a harbinger of better things to 
come with respect to elucidation of radiation effects on cell organelles, is 
the inhibition of change in fatty acid ratios of nuclei in all irradiated rats. 

The changes in nuclear fatty acid ratios that occur normally as a function 
of age have tentatively been interpreted as reflecting an advance in the ma- 
turity of these organelles. Thus, one of the interpretations for the inhibition 
of these shifts in ratios following irradiation is that maturation (or diflFeren- 
tiation) of the nucleus has been retarded due to injury by oxidizing radicals. 
However, in a heterogenous tissue, such as brain, changes in organelle fatty 
acid ratios may merely reflect changes in the proportions of resident cells. 
The importance of good histology as an adjunct to biochemical studies in 
these tissues is thus emphasized. 

In most animals receiving irradiatic^n to the head only, there was a de- 
crease in total body weight and the weights of such organs as liver, spleen, 
kidney and heart. Although the changes in weights of the various brain 
components did not appear to correlate closely with the weight changes of 
the above organs, the influence of irradiation on the pituitary (and hence a 
probable change in output of certain hormones) is a possible factor in- 
fluencing the observed results. However, a personal communication from Dr. 
Van Dyke, of the University of California at Berkeley, indicated that growth 
hoiTnone administered to irradiated neonatal rats has no effect in delaying 
the onset — or modifying the intensity — of neurological aberrations. 

Summary 

All three major divisions of the brain (brain stem, cortex, and cere- 
bellum) were inhibited in growth following irradiation (750 r) to the head 
at 2 days of age. Growth of brain stem was not retarded until about the 16th 
day, and due to a relatively slow rate of growth in the control animals, the 
difference in dry weight of this part of the brain at 4 weeks postirradiation 
was not great. In corte.x. the inhibition of growth was also first discernible 
at about 16 days, but due to a relatively fast rate of increase in the control 
animals, the differential between control and irradiated cortices at 4 weeks 
was very significant. Cerebellum was most profoundly affected by x-irradia- 
tion, the decrease in size being quite apparent early in the 2nd week follow- 
ing exp)csure. 



108 SCHJEIDE, YAMAZAKI, CLEMENTE, RAGAN AND SIMONS 

Although in this preliminary survey no difTerences could be detected be- 
tween irradiated and control cortices (and cerebellums) in terms of relative 
lipid, relative phospholipid, and relative nitrogen, irradiated brain stem 
apf)eared to contain relatively less total lipid beginning at about 14 days fol- 
lowing exposure. The inhibition of lipid synthesis in the brain stem (failure 
of myelination) was accompanied by a parallel retardation of the dehydra- 
tion normally occurring as a function of age. 

Some tissues from irradiated brain revealed decreases in wet volumes of 
mitochondria. In no case was there a significant increase of mitochondria in 
irradiated tissue. 

The ratio of palmitic acid to stearic acid and to oleic acid in the nuclei 
decreased as a function of age. The de\elopment of this ratio was retarded 
in the irradiated tissues examined. 

References 

Folch-Pi, J. 1955. Composition of brain in relation to maturation. In "Biochemistry 
of the Developing Nervous System" (H. Waelsch, ed.), pp. 121-136. .Academic 
Press, New York. 

Gal, E. M., Fung, F. H., and Greenberg, D. M. 1952. Studies on the biological 
action of malononitriles, II. Distribution of rhodanese ( transulfurase) in the tissues 
of normal and tumor-bearing animals and the eflPect of malononitriles thereon. Can- 
cer Research 12, 574-579. 

Hicks, S. P. 1952. Some effects of ionizing radiation and metabolite inhibition on 
the developing mammalian nervous system. /. Pediat. 40, 489. 

Russell, L. B. 1954. Effects of radiation on mammalian prenatal development. In 
"Radiation Biology," (A. Hollaender, ed.). Vol. I, pp. 861-918. McGraw-Hill, 
New York. 

Schjeide, O. A. 1960. Unpublished data. 

Schjeide, O. A.. Ragan, N.. McCandless. R. G.. and Bishop, F. C. 1960. Effect of 
x-irradiation on cellular inclusions In chicken embryo livers. Radiation Research 
13, 205-213. 

Sperry, W. M. 1955. Lipid analysis. Methods of Biochem. Anal. 2. 83-111. 



Some Effects of Nucleic Acid 
Antimetabolites on the Central Nervous 
System of the Cat* 



Harold Koemg 

Veterans Administration Research Hospital and 

Xorthuestern University Medical School. 

Chicago, Illinois 



Amont; the biolooic effects imputed to ionizini; radiation is a disturbance 
in nucleic acid metabolism. The deleterious effect of ionizin" radiation on 
DNA. particularly in proliferating tissues, is well known (Seed. 1960 i. Its 
influence on RNA metabolism has received less attention, lliat it is not 
without effect on the latter. howe\er. is suijgested by several recent studies 
(Krogh and Bersjeder. 1957; Schummelfeder. 1957). It may be sci'mane to 
this symposium to describe some of the effects of certain nucleic acid anti- 
metabolites on the mammalian central nervous system. 

These studies had their inception in obser\ations made earlier with the aid 
of taooed precursors which showed that neurons, olioodendroolia. and certain 
other cells are site of acti\e RNA and protein turno\er i Koeni',;. 1958a,b). 
A slow labeling of DNA also occurs among these cells, neurons excepted, 
which probably indicates cell di\ision. We ha\e attempted to interlere with 
these metabolic activities through the use of nucleic acid antimetabolites. 
Intrathecal administration was used to circum\ent the blood-brain barrier 
and to attain adequate local concentration of antimetabolities in the nervous 
system without damaging hematopoietic and other susceptible tissues. Of many 
purine and pyrimidine analogs tested, several ffuorinated pyrimidines were 
tound to produce interesting neurologic disorders i Koenig, 1958ci. The 
neurotoxic antimetabolites were 5-fluoroorotic acid i FO i , the analog of 
orotic acid > the natural precursor of pyrimidines i and the ribosides. 5-fluoro- 
uridine FUR) and 5-fluorocytidine FCR ) (Fig. 1 i. The pyrimidine bases, 
5-fluorouracil • FU ) and 5-fluorocytosine i FC ) , were without overt effect, 
even in large doses. The clinical, pathologic, and biochemical effects of these 
analogs, particularly FO. on the feline neuraxis ha\e been imder inxestiga- 
tion for several years. .Although their biochemical effects ha\e not been 

* Supported in part by grants from The U.S. Public Health Service and the .Atomic 
Energy Commission. 

109 



110 







HAROLD 


KOENIG 




OH 

1 








OH 


A i 

HO^ ^N-^ 


^COOH 






HO ^N ^CO( 


Orotic ac 


id 






5-Fluoroorotic 
acid (FO) 



Fig. 1. Structural formulas of orotic acid and 5-fluoroorotic acid. 



worked out completely, sufficient data have been collected to warrant the 
conclusion that the neurologic eflfects of these compounds are attributable, 
directly or indirectly, to disturbances in pyrimidine nucleotide or nucleic 
acid metabolism or both. 

Intracisternal Administration 

Intracisternal administration of 5-15 mg of sodium salt of FO (or 2-5 mg 
FUR) in cats produces a progressive rhombencephalopathy and, in some 
animals, a cervical myelopathy ( Koenig, 1958c). Signs of nemal dysfunction 
appear on the 4th to 6th day after injection. The first indication of disease is 
a mild clumsiness of gait, which worsens in time. The gait becomes broad- 
based, unsteady and dysmetric. Decomposition of movement, oscillation of 
trunk and limbs, and reeling gait complete the picture of cerebellar ataxia. 
This usually becomes so disabling that the animal is incapable of locomotion 
or alimentation by the 2nd or 3rd week after injection of the antimetabolites. 
Many animals have signs of neuronal irritation, including fasciculations of 
facial musculature, myoclonic jerks of lorelimbs, and various tonic and 
runnings seizures. Animals die of inanition, seizures, or bulbar failure by the 
3rd week. Outstanding pathologic distmbance is a depletion of Nissl sub- 
stance in Purkinjc neurons of the cerebellum and in neurons of the brain 
stem and cervical spinal cord. 

Intraspinal Injection 

Injection of FO into the lumbar subarachnoid space ( 10-15 mg divided 
into two doses and injected 3-4 hours apart ) in cats produces a progressive 
myelopathy, which becomes evident on the 2nd or 3rd day (Koenig, 1960). 
FUR and FCR produce a similar disorder in doses of 2-4 mg. Signs of 
neuronal irritation appear first. These consist of muscle fasciculations, hyper- 
esthesia, and sometimes myoclonic jerks in the hindquarters (Fig. 2) and 
are associated with clumsiness, mild weakness, enhanced stretch reflexes, and 



NUCLEIC ACID ANTIMETABOLITES AND CNS 111 



J^loo// V I— I = 25 m^ed. 



Fig. 2. Fasciculations recorded electromyographically in the hamstrings 3 days 
after FO. 



hypertonia of flexor muscles in the hindlimbs. In some animals this may not 
progress beyond a hyperreflexic paraparesis. In more severely affected 
animals, the signs of neuronal irritation diminish and paraplegia with loss 
ot muscle tone and stretch reflexes ensues. Sensation becomes obtunded, and 
the sphincters are paralyzed. These signs indicate a loss of neuronal function. 
Denerxation atrophy with fibrillary potentials may appear in the 2nd week, 
presumablv because some motoneurons are destroyed. Animals with a mild 
myelopathy, i.e.. a hyperreflexic paraparesis, after a period of stability or 
improvement enter a second stage of illness during the 3rd to 4th week. 
Their condition worsens, and within a day or two they exhibit an extensor 
paraplegia with dulling of sensibility in the hindquarters and acute urinary 
retention. Spastic weakness of the forelimbs sometimes occurs later. In 
most severely afflicted animals, flaccid paraplegia appears in 4 to 5 days. The 
forelimbs are affected early, and death occurs from respiratory failure by 5 
to 7 days. In general, the larger the dose of the analog, the more severe is the 
myelopathy. 

The histopathology of the myelopathy produced by FO has been carefully 
in\estigated ( Koenig, 1960). The changes initially are confined to neurons. 
Inflammatory or vascular lesions do not occur. The nucleoli of nerve cells 
become small and less basophilic. Within 3 or 4 days a fragmentation and 
loss of peripherally situated Nissl substance is seen in spinal motoneurons 
and interneinons i Fig. 3 ) . Depletion of Nissl substance progresses to involve 
much of the perikaryon by 7 to 10 days i Fig. 4). Signs of recovery then ap- 
pear in \iable neurons. These consist of a striking hypertrophy and an in- 
crease in basophilia of nucleoli i Fig. 5 ) followed by increasing amounts of 
Nissl substance. Regeneration is well ad\anced by 35 to 50 days, and some 
neurons are even chromophilic. White matter is structurally intact for 2 to 3 
weeks, but thereafter it almost always exhibits some spongy or mycrocystic 
degeneration with \arying Cjuantities of neural fat, either free or in macro- 
phages I Fig. 6 j . A thinning of oligodendroglia is seen in white matter at this 



112 



HAROLD KOENIG 






Fig. 3. Peripheral chromatolysis with reduction in size and basophiha of nucleoli 
in neurons of L-7 three days after FO. Thionin, X 700. 

Fig. 4. Generalized chromatolysis of lumbar motoneuron seven days after FO. 
Gallocyanin, X 800. 




Fig. 5. Recovering lumbar motoneuron 21 days after FO. Note hypertrophy and 
increased basophilia of nucleolus. Gallocyanin, X 700. 



stage. The white matter lesion occurs at a time when neurons are recovering 
and probably results from oligodendroglial disease. 

Signs of neuronal irritability are correlated with a minimal to moderate 
depletion of Nissl substance. Loss of neuronal function is associated with 
severe loss of Nissl substance. In most severelv affected animals, a necrosis of 



113 



Fig. 6. White matter, L-1, 50 days after FO. Note status spongiosiis. Weil stain, 
X 195. 



gray matter appears early, but this is not common unless 20 ma, or more of 
FO are used. The delayed, subacute deterioration in spinal cord function is 
associated with spongy deseneration of white matter. 



Intracerebral Administration 

The injection of 4-6 mg FO i 2-3 ms: FUR) into the sensorimotor cortex 
of the cat (3-5 mm imder the pial smface through a 26 gauge needle) pro- 
duces a focal cortical encephalopathy i Kurth (7 al., 1960). A moderate 
hemiparesis, propriocepti\e deficit, and impaired contact placing reaction 
appear in the contralateral limbs 3 or 4 days after injection. Focal motor 
seizures are sometimes seen. The disorder progresses for several davs and 
then becomes stationary or impro\es slightly. Neural dysfunction persists for 
several months. A focal electroencephalographic defect is demonstrable in 
the \icinity of the injection. Normal ihythms are replaced in part by slow 
wa\es, sharp waxes, and high \oltage spikes ( Figs. 7 and 8 ) . Focal seizure 
actixity appears spontaneously or may be provoked by joint movement or 
skin pinching of the contralateral limbs. Biochemical studies ha\e rexealed a 
high incorporation of FO-2-C" into RNA near the injection site. Indeed, 
there is a close correlation between the presence of electroencephalogiaphic 
abnormalities and a high uptake of FO into RNA of affected cortex. 

The introduction of FO or FUR into the temporal lobe of cats may pro- 
duce alterations in personality and epileptiform seizures (Koenig ct al., 
1960bi. Some animals become withdrawn, imfriendly. hostile, and e\en 
aggressive, with periods of confusion, stupor, and other disturbances of be- 
havior indicative of temporal lobe seizures. Sometimes the contralateral pupil 



114 



HAROLD KOENIG 



Rt. frontal lobe 




Transverse frontal 



Vl 



Fig. 7. EEG 16 days after injection of 6 mg FO into right frontal lobe. Note 
numerous spikes. Pentobarbital anesthesia. 




Mit¥ 



Rl. fronUl loll,. 



V/'-'v^vv-i 



Ll. frontal lobe 




/^"^'L^l'l' 



Fig. 8. EEG 15 days after injection of 6 mg FO into right frontal lobe. Note 
paroxysm of high voltage activity. Pentobarbital anesthesia. 



beconie.s dilated, and the nictitating membrane retracts. Focal electroen- 
cephalographic defects appear 2 to 4 days after injection, with slow waves, 
sharp waves, and spikes (Figs. 9 and 10). Some of these abnormal rhythms 
are propagated to the opposite temporal lobe. Spontaneous focal and general- 
ized electrical seizures occur, even though animals are under pentobarbital 
anesthesia. Cytopathologic changes occur in cortical neurons which are simi- 
lar in character to those observed when FO is administered elsewhere in the 
central nervous system. However, the changes are mild and often unrecog- 
nizable when neuronal dysfunction is present. 



NUCLEIC ACID ANTIMETABOLITES AND CNS 



115 








= s 



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Metabolism of FO 



The metabolism of taoged FO (FO-2-C") in the spinal cord of cats has 
been inxestigated by autoradiographic and biochemical methods (Koenig 
and Young, 1960; Koenig et al., 1960a). Column and paper chromatography 
have been used for nucleotide analysis. FO is metabolized similarly to orotic 



116 



HAROLD KOEiNIG 

_h h K. 




Rt. temporal lobe 



^MA/n/j/i.v'Y^ 



Ll. temporal lobe 




Transverse temporal 

Fig. 10. EEG 6 days after 12 mg FUR into right temporal lobe. Note seizure ac- 
tivity. Pentobarbital anesthe.sia. 



acid, the natural precursor of pyriniidiiies in the nervous system. These 
studies have disclosed that FO is efficiently converted into the followina, acid- 
soluble nucleotides in nervous tissue: fluorouridine monophosphate, diphos- 
phate, and triphosphate: fluorouridine diphosphate-2,lucose and diphosphate- 
acetyl2,lucosamine, and fluorocytidine monophosphate (Fig. 11). FO is in- 
corporated into RNA as fluorouridine monophosphate, but is not incorporated 
into DNA. Unlike FO, 5-fluorouracil (FU), is not conxerted efficiently into 
acid-soluble and RNA nucleotides in the feline neiuaxis. Uracil itself also is 
poorly metabolized by this tissue. 

These observations suggest that nucleoside phosphorylase, the enzyme 
which converts pyrimidine bases to their ribosides, is present in scanty 
amounts in the central nervous system of the cat. Poor anabolic conversion 



NUCLEIC ACID ANTIMETABOLITES AND CNS 



117 



PUMP 
♦•PCDP 



FCMP 




70 



FUDP 
+FCTP 



POMP 
i FUDPAG 
.FUDPG 




FUTP 



100 150 200 

Tube No. 



2 50 



300 



Fig. 11. Chromatogram of acid-soluble fraction of spinal cord obtained by extended 
gradient elution with formic acid-ammonium formate from Dowe.x 1 column. Tissue 
removed 4 hours after the intraspinal injection of FO-2-C". 

Key: — D 260 um: Radioactivity of C". 



of FU and FC probably accounts for failure of these analogs to produce 
neurologic distiubances. Similar anabolic conversions of these fluorinated 
pyrimidines ha\e been described in other organs i Harbers ct al., 1959). 

The morphologic distribution of incorporated FO has been e.xamined by 
high resolution autoradiography ( Koenig and Young. 1960). FO is taken up 
into RNA of neurons and oligodendroglia and leptomeningeal. ependymal, 
and Schwann cells. Initially FO is incorporated into nuclear RNA. Some 
labeling of cytoplasmic RNA is discerned in nerve cells after a day or so; 
however, FO persists in nuclear RNA for a number of weeks. 



Metabolic Effects of FO 

The histopathologic changes produced by FO suggest a depletion of 
neuronal RNA. The results of biochemical analysis corroborate this inference 
(Koenig ct ai, 1960a) . The concentration of RNA in gray and white matter 
of spinal cord diminishes by 30-50% after 1 week. A greater depression in 
RNA concentration occurs in animals with areflexic paraplegia than in those 
with hyperactive reflexes. The incorporation of labeled orotic acid and 



118 HAROLD KOEMG 

adenine into RNA in vivo is depressed by FO (Fig. 12). FO evidently inter- 
feres with the biosynthesis of RNA in neural tissue. The mechanism by which 
FO brings about a depletion of RNA, however, has not been ascertained at 
the time of this writing. 

The well-known participation of RNA in protein synthesis has led us to 
investigate the uptake of labeled amino acids into protein. Initially, FO does 
not depress the incorporation of tagged methionine and lysine into neural 
protein. A depression of 50-85% is observed after 1 week, however. Signifi- 
cantly, the greatest depression in uptake is observed in cases of severe neu- 
ronopathy, i.e., when areflexic paraplegia is present. Thus, a loss of neuronal 
function is associated with a greater depletion of RNA and a severe defect 
in protein biosynthesis in afTected nerve cells (Fig. 13). The formation of 
spurious RNA molecules also could contribute to the defect in protein 
synthesis. 

The role of pyrimidine nucleotides as cofactors in lipid and polysaccharide 
biosynthesis and in interconversion of sugars has been recognized recently 
(Henderson and LePage, 1958). The formation of spurious fluoropyrimidine 
nucleotides suggests that disturbances in lipid and carbohydrate metabolism 
may be partly responsible for the neurologic disorders that are produced by 
the fluorinated pyrimidines. This possibility is being investigated. The bio- 
chemical basis for the neuronal hyperirritability also remains to be elucidated. 



If- 




■4 




Fig. 12. Autoradiographs showing depressed uptake of orotic-6-C" into RNA of 
lumbar motoneuron 2 days after FO. Control on left, experimental on right. X 700. 



NUCLEIC ACID ANTIMETABOLITES AND CNS 



119 



Summary > ? .• < 

Intrathecal administration of the fluorinated pyrimidines, FO. FUR, and 
FCR, results in interesting neurologic disorders, the nature of which depends 
on the injection site. Myelopathy, rhombencephalopathy, and cortical en- 
cephalopathy are produced by the intralumbar, intracisternal, and intracere- 
bral routes, respectively. An asymptomatic "incubation'' period precedes the 
appearance of neural dysfunction. Signs of neuronal hyperirritability ap{3ear 







*^«• 



J ■• 






* D FO HYPERREFLEXIC P.\RAPARESIS 



■■'■ 


.■■••.' 




jk s ^Bfl^^^^B.* * 




t-^B^^^KKBK^^^^ * 




^p,. 


£: 


■ - 



'■#* 



7 D FO in'PERREFLEXIC PARAPARESIS 7 D FO HYPOREFLEXIC PARAPLEGIA 



Fig. 13. .Autoradiographs of L-7 segment showing uptake of methionine-S"^ into 
neuronal protein. Note reduction in blackening over experimental neurons three and 
seven days after FO, most marked in animal with hyporeflexic paraplegia. X 700. 



120 HAROLD KOENIG 

first. In more severe intoxications, loss of neuronal function follows the stage 
of neuronal irritation. Alterations in neuronal structure accompany the 
neurologic disorder. RNA-containing structures, i.e., nucleoli and NissI 
bodies, are conspicuously aflfected. The neuronopathy may be reversible or 
may result in necrobiosis, depending on the severity of intoxication. Spongy 
degeneration of white matter occurs later, probably caused by oligoden- 
droglial disease. 

FO undergoes conversion to acid-soluble nucleotides and is incorporated 
into RNA in the feline neuraxis. Indeed, anabolic con\ersion of the fiu- 
orinated pyrimidines seems to be a requirement for the production of neural 
dysfunction. A depletion of RNA and a depression in protein biosynthesis 
appear later and are most marked in neurons that become inexcitable. Dis- 
turbances in other metabolic spheres may e.xist, but have not been demon- 
strated. Many points of similarity, both physiologic and pathologic, can be 
discerned between the disorders described and some virus infections and 
degenerative diseases of the nervous system. It seems possible, therefore, that 
derangements in pyrimidine nucleotide or nucleic acid metabolism may be 
present in some of the latter disorders. The fiuorinated pyrimidines are 
useful tools for the production of focal neuronal disease. Their use may 
provide experimental models for some of the degenerati\e neuronal diseases 
that afflict man. 

References 

Harbcrs, E.. Chaudhuri, N. K.. and Heidtlberger, C. 1959. Studies in fluorinated 

pyrimidines: VIII. Further biochemical and metabolic investigations. /. Biol. 

Chem. 234, 1255-2162. 
Henderson, J. F., and LePage, G. .\. 1958. Naturally occurring acid-soluble nucleotides. 

Chem. Revs. 58, 645-687. 
Koenig, H. 1958a. Incorporation of adenine-8-C" and orotic-6-C" acid into nucleic 

acids of the feline neuraxis. Proc. Soc. Expl. Biol. Med. 97, 255-260. 
Koenig, H. 1958b. An autoradiographic study of nucleic acid and protein turnover in 

the mammalian neuraxis. /. Biophys. Biochem. Cytol. 4, 785-792. 
Koenig, H. 1958c. Production of injury to the feline central nervous system with a 

nucleic acid antimetabolite. Science 127, 1238-1239. 
Koenig, H. 1960. Experimental myelopathy produced with a pyramidine analogue. 

A.M.A. Arch. Neurol, 2. 463-475. 
Koenig, H. and Young, I. J. 1960. .Autoradiographic studies of nucleoprotein metab- 
olism in the neuronopathy produced by a pyrimidine analog. Anat. Record 136, 

224. 
Koenig, H., Gaines, D., Wells, W., Young, I. J., and Muniak, S. 1960a. Unpublished 

data. 
Koenig, H., Young, I. J., and Kurth, L. E. 1960b. Unpublished data. 
Krogh, E. v., and Bergeder, H. D. 1957. Experimental irradiation damage of the 

cerebellum demonstrated by gallocyanin-chromalum staining method. /" Cong. 



NUCLEIC ACID ANTIMETABOLITES AND CNS 121 

intern. Sci. Neurol., Brussels, 1957: 3' Congr. iniern. Neuropathol. pp. 287-294. 

Acta Medica Belgica, Brussels. 
Kurth, L. E., Koenig, H., and Freyre. J. 1960. Frontal lobe encephalopathy with 

focal seizures produced with pyrimidine analogs. Trans. Am. Neurol. Assoc. 85, 

215-216. 
Schiimmelfeder, N. 1957. Fluoreszenzniikroskopische und cytochcmische untersuchun- 

gen iiber Friihschaden am Kleinhirn der Maus nach Rontgenbestrahlung. /"" Congr. 

intern. Sci. Neurol, Brussels, 1957: .1' Congr. intern. Neuropathol. pp. 295-308. 

Acta Medica Belgica, Brussels. 
Seed, J. 1960. Inhibition of nucleic acid synthesis caused by x-irradiation of the 

nucleolus. Proc. Roy. Soc. B152, 387-395. 



Geographic Distribution of Multiple 
Sclerosis in Relation Geomagnetic 
Latitude and Cosmic Rays* 

John S. Barlow 

Massachusetts General Hospital, 
Boston, Massachusetts 



Introduction 

Despite the fact that multiple sclerosis has been known as a clinical 
entity for over a century, its etiology is still an enigma (Schumacher, 1960). 
Moreover, there is no specific treatment for the disease, which in its later 
stages often results in severe crippling. The disability results from interfer- 
ence with the processes of electrical conduction along nerve fibers in the 
brain and spinal cord as the myelin sheath of the fibers degenerates in local- 
ized regions; the term "demyelinating disease" is accordingly used. 

One of the interesting aspects of the disease is its geographic distribution. 
It has been a clinical impression for some years (Steiner, 1938) that multiple 
sclerosis does not have a uniform distribution throughout the world, and 
several epidemiologic surveys have been undertaken to clarify this distribu- 
tion (McAlpine et al., 1955: Hyllested. 1956: Kurland ct ai, 1957). The 
disease appears to be appreciably more common in northern than in southern 
latitudes in North .America and in Europe, but uncommon in the Orient, 
South America. Africa, and the tropics and subtropics. 

There have been several possible explanations ad\anced for this geo- 
graphic distribution, some of which I have re\iewed elsewhere (Barlow, 
1960), but none has appeared to be consistent with all of the a\ailable 
data for the distribution. More recently, Acheson et al. (1960) have found 
that the geographic distribution of multiple sclerosis among veterans in the 
United States correlates strongly in an inverse manner with the average solar 
radiation of place of birth, and in particular with the December solar radia- 
tion, the implication being that this agent may in some way act as a preven- 
tive or protective agent against multiple sclerosis. When the distribution by 
residence at onset of symptoms was examined, the correlation appeared best 

* This work was supported by the National Institute of Neurological Diseases and 
BHndnesSj U.S. Public Health Service. 

123 



124 JOHN S. BARLOW 

with geographic latitude. Since, according to these authors, the isolines for 
winter solar radiation follow the lines of geographic latitude fairly closely, it 
is not clear that the relationship observed between December solar radiation 
and distribution of multiple sclerosis by birthplace would prevail in a similar 
manner for other areas of the world, for geographic latitude itself does not 
appear to be a good correlate when data for the disease from different areas 
of the world are examined (Barlow, 1960). It may well be, however, that 
the geographic distribution of the disease is deteiTnined by several factors, 
and the importance of each of these may vary in different areas. 

The possibility that geologic factors, perhaps in trace elements, may have 
a role in the distribution of multiple sclerosis recently has been reiterated by 
Warren (1959). 

Latitude Distribution of Multiple Sclerosis 

In the United States in recent years. Dr. Leonard T. Kurland of the Epi- 
demiology Branch of the National Institute of Neurological Diseases and 
Blindness has been particularly concerned with surveys on multiple sclerosis, 
and it was as a result of his epidemiologic summary at the First International 
Congress of Neurological Sciences in Brussels in 1957 that the present ap- 
proach had its origin. At that congress, Kurland presented data for North 
America which indicated that the frequency of the disease is strongly 
dependent on latitude, and he suggested that any satisfactory explanation of 
the etiology must take the geographic factor into account (Kurland et al., 
1957), a view also held by other observers (McAlpine et al., 1955). 

The variation of the disease with latitude particularly interested me, and I 
undertook to determine if there were any similarity between this latitude 
effect and the intensity of cosmic rays, whose distribution is well known to 
be dependent in part on latitude (Barlow, 1959). 

The variation of cosmic ray flux is determined by the earth's magnetic field 
and therefore is related to geomagnetic latitude rather than to geographic 
latitude. A map indicating geomagnetic latitude in relation to geographic 
latitude is reproduced in Fig. 1. The lines of constant geomagnetic latitude 
are skewed with respect to those of constant geographic latitude, derived 
from the fact that the earth's magnetic axis is inclined at an angle of about 
10° with respect to its axis of rotation. For the eastern United States, the 
geomagnetic latitude for a given location is about 10° greater than its geo- 
graphic latitude; for western Europe, the two are approximately the same, 
and for eastern Asia, the geomagnetic latitude for a particular location is 
about 10° less than its geographic latitude. It is apparent then that the two 
latitudes may differ from one another by an amount up to plus or minus 10°, 
a total rans;e of about 20°. 



GEOGRAPHIC DISTRIBUTION OF MULTIPLE SCLEROSIS 



125 



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Prevalence Surveys 

To study the latitude effect for multiple sclerosis, several independent sets 
of statistics for the United States and other areas in the world were examined 
(Barlow, 1960). These included mortality data, prevalence data, (i.e. data 



126 



JOHN S. BARLOW 



concerning the total number of cases in a population at a particular time), 
incidence data (i.e. the number of new cases appearing in a population per 
year), and statistics concerning hospital admissions for the disease. 

Since multiple sclerosis is a chronic disease (the average case has a dura- 
tion of approximately 20 years after onset of symptoms) , it appears that the 
most reliable indication of its distribution may be obtained from prevalence 
data. Table I shows results from a series of surveys in the United States and 

TABLE I 

Multiple Sclerosis Prevalence Ratios for the White Population 
IN Selected Communities in the United States and Canada" 















Prevalence per 










Geogra 


phic 


Geomagnetic 


100,000 


Prevalence rel- 


City 




latitude 


rN) 


latitude 


CN) 


population '' 


ative 


' to Winnipeg 


Winnipeg 




50 




60 




42 (40) 




1.0 (0.95) 


Boston 




42 




53 




41 




0.97 


Denver 




40 




48 




38 




0.90 


San Francisco 


37 




43 




30 




0.71 


Charleston, 


S. 


C. 32 




43 




18 (12) 




0.43 (0.29) 


New Orleans 


30 




40 




13 (6) 




0.31 (0.14) 



" Data from Kurland el at., 1957. 

* Values in parentheses are corrected values on "clinical review" of the reported cases. 



Canada. In the present study, emphasis is laid on relative rather than abso- 
lute frequencies of occurrence of the disease in diflferent areas, and the table 
indicates the prevalence in each city relative to that for Winnipeg. Relative 



1.0 



0.8 



0.6 






0.2 - 



OL- 




20 30 40 50 30 40 50 60 

Geographic latitude °N Geomagnetic latitude °N 

Fig. 2. Relative prevalence of multiple sclerosis in selected communities in the 
United States and Canada (see Table I). 



GEOGRAPHIC DISTRIBUTION OF MULTIPLE SCLEROSIS 127 

prevalence is plotted against geographic and geomagnetic latitude in Fig. 2, 
and the points suggest a sigmoid curve. A normal sigmoid curve (i.e., the 
integral curve for a normal distribution) has been included in each plot, and 
it is evident that it is not possible to conclude from these data drawn from a 
limited range of longitude whether geomagnetic latitude is a better param- 
eter than geographic latitude. The general trend shown by the points 
between approximately 40° and 50° has been confirmed from several other 
types of data for the United States (Barlow, 1960; Acheson, 1959) , although 
the presence of a "knee" at about 50° geomagnetic latitude is not clear from 
these latter data. Evidence that a rapid rise in the frequency of the disease 
between geomagnetic latitudes of approximately 40° and 50° appears for 
other areas of the world in both the northern and southern hemispheres has 
previously been presented (Barlow, 1960). 

For further examination of the latitude distribution, prevalence data from 
recently conducted surveys presented at the Geomedical Conference in 
Copenhagen in June, 1959 (Hyllested, 1960) are listed in Table II. It 
also includes the data from Table I and results of other prevalence surveys. 
As in Table I, the mean prevalence ratio for locations of 50° or greater geo- 
magnetic latitude was determined, and prevalences relative to this standard 
(48 cases per 100,000) are shown. These data are plotted against geographic 
and geomagnetic latitude in Fig. 3. Since a wide range of longitudes is repre- 
sented in these plots, the sigmoid curve for the geomagnetic plot of Fig. 2 is 
reproduced in both the geographic and the geomagnetic plot of Fig. 3, and 
it will be reproduced in subsequent plots for comparative purposes. 

It is apparent from inspection of the two plots that at any given latitude the 
scatter of the p>oints is greater for the geographic plot than for the geo- 
magnetic plot, at least for latitudes of less than 50°. The scatter of the points 
above 50° is such that a "knee" or leveling off is not as clearly suggested as 
in Fig. 2. There is some indication, however, that the rapid increase of 
prevalence between 40° and 50° geomagnetic latitude does not continue 
upward in the same manner beyond 50°. 

Several independent surveys are represented in Fig. 3; therefore it is not 
possible to state how much of the scatter of points above 50° geomagnetic 
latitude is due to differences in survey procedures and how much is due to 
real differences in prevalence among the population groups. It is probably 
unlikely that the scatter is entirely due to differences in survey procedures. 
Even if uniform survey procedures were used, such a scatter of points could 
conceivably occur if the prevalence ratios among different population groups 
formed a normal distribution about a mean value, and the scatter might 
further be accentuated by differences in the size of the population groups. 
Particularly if the population is very small, chance variations in the observed 
prevalence ratios may be pronounced for occasional communities (Deacon 
et al, 1959). 



128 



JOHN S. BARLOW 



TABLE II 

Prevalence of Multiple Sclerosis at Various Latitudes 





<o 


/o 


■a, -"^ 


^ 










to 


.li 




City or country 


« 3 


2 .^ 

1 3 


an ^ 


II 

•a. 


Investigator 


Surveys des( 


;ribed at the 


Geomedical 


Conference " 


Faeroe Islands 


62 


65 


19 


0.40 


Fog (1960) 


West Norway 


60 


61 


17" 


0.36 


Presthus (1960) 


East Norway 


60 


60 


37" 


0.77 


Oftedal (1960) 


Goteburg, Sweden 


58 


57 


62 


1.3 


Borman (1960) 


Denmark 


56 


56 


64 


1.3 


Hyllested (1960) 


Durham & North- 












umberland Coun- 












ties, England 


55 


57 


54 


1.1 


Miller (1960) 


Gronigen, Holland 


53 


54 


56 


1.2 


Dassel (1960 


Spessaert, Germany 


50 


51 


75 


1.6 


Bammer and 
Schaltenbrand 
(1960) 


Switzerland 


47 


47 


50 


1.0 


Georgi and Hall 
(1960) 


Missoula, Montana ■* 


47 


55 


55 


1.3 


Siedler^«a/. (1958) 


Halifax, Nova Scotia " 


46 


56 


32 


0.70 


Alter et al. (1960) 


Sapporo, Japan 


43 


33 


1.6 


0.04 


Okinaka et al. 
(1960) 


Turkey 


39 


35 


2.5 


0.05 


Mutlu (1960) 


Charleston, 












South Carolina '' 


33 


44 


14 


0.30 


Alter et al. (1960) 


Kumamoto, Japan 


33 


21 


1.8 


0.04 


Okinaka et al. 
(1960) 


Egypt 


27 


24 


0.2 


0.004 


Georgi and Hall 
(1960) 


Yemen 


15 


11 


'Unknown" 


— 


Georgi and Hall 
(1960) 


East Africa 


-10 


-6 


"Very few" 


- — ■ 


Georgi and Hall 
(1960) 




Surveys cited by Kurland et al. 


(1957) 




Northern Scotland 


58 


61 


55 


1.1 




Northern Ireland 


55 


58 


41 


0.86 




Rochester, Minnesota ■* 


44 


53 


64 


1.3 




Kingston, Ontario * 


44 


55 


53 


1.1 




Winnipeg, Manitoba ' 


50 


60 


40 


0.83 




Boston, Massachusetts ' 


42 


53 


41 


0.86 




Denver, Colorado ^ 


40 


48 


38 


0.80 




San Francisco "^ 


37 


43 


30 


0.63 




New Orleans " 


30 


40 
Oth 


6 

er surveys 


0.13 




Budapest 


47 


46 


20 


0.42 


Halasy (1957) 



GEOGRAPHIC DISTRIBUTION OF MULTIPLE SCLEROSIS 



129 




10 20 30 40 50 

CEOGBAPmC LATITUDE ^N GEOMAGNETIC LATITUDE "N 

Fig. 3. Relative pre\alence of multiple sclerosis for the localities listed in Table II. 
The sigmoid curve included in this and in subsequent figures is reproduced from that 
for the geomagnetic plot in Fig. 2. 

Two surveys included in Table II are of special interest, for they were 

conducted at different latitudes in areas with almost the maximum possible 

range of difference between geographic and geomagnetic latitudes, i.e., in 

North America and Japan. Moreo\er, each of the two sur\eys included the 

1.0 r- + 



0.8 



0.6 



0.4 



0.2 



OL- 




30 40 50 60 20 30 40 50 60 

Geographic latitude °N Geomagnetic latitude °N 

Fig. 4. Relative prevalence of multiple sclerosis for four communities in North 
America and Japan (see Table III). 



Footnotes to Table II on Facing Page 128 
"1.0 corresponds to the mean prevalence for 50 or greater geomagnetic latitude (48 per 100.000). 
l" Hyllested, K. (ed.) 1960. Report on the Geomedical Conference in Copenhagen, 1959. Studies in 
Multiple Sclerosis III. Ada Psychiat. Neurol. Scand. 35, Suppl. 147, 158 pp. 
<^ Provisional. 
1 U.S.A. ' Canada. 



130 



JOHN S. BARLOW 



ct; 



o 



«,-« 



o 



^ ^ Tj- 



.5 o 



< O 



GEOGRAPHIC DISTRIBUTION OF MULTIPLE SCLEROSIS 



131 



same team of observers for the two latitudes studied and similar diagnostic 
criteria (Alter et al., 1960; Okinaka et al., 1960). Additional details for 
these two surveys are shown in Table III, with prevalences relative to that 
for Halifax. Plots for this table are reproduced in Fig. 4, and it is evident 
that for these two surveys the geomagnetic coordinate provides a much better 
parameter than does the geographic coordinate, a conclusion not appreciably 
altered by assumption of a reference prevalence of 33.6 per 100,000 popula- 
tion instead of the 48 per 100,000 of Fig. 3 or of 42 per 100,000 of Fig. 2. 
Moreover, it is apparent that the sigmoid curve in the previous geomagnetic 
plots is also a good fit for this plot in Fig. 4. 

Multiple Sclerosis in the Soviet Union 

Because of its considerable geographic extent, data on the occurrence of 
multiple sclerosis in the Soviet Union would be of considerable interest. 
Grashchenkov and his associates (1960) have recently investigated the mor- 
bidity due to this disease in different regions of the Soviet Union by com- 
parison of the percentages of cases of multiple sclerosis in relation to the 
total number of patients of sei^vices of neurology during 1948-1957 (Table 
IV) . Such data on the relative frequency of a disease are not directly com- 
parable with mortality statistics or prevalence data for other areas of the 



1.2 



1.0 



k 

K 0.2 



0.8 



0.6 



0.4 



0.0 




X-K) 



X-*o 




30 40 50 60 70 

GEOGRAPHIC LATITUDE 'N 



20 30 40 50 60 70 

GEOMAGNETIC LATITUDE 'N 



Fig. 5. Relative number of hospital admissions for multiple sclerosis in selected 
communities in the Soviet Union (see Table IV). 



132 



JOHN S. BARLOW 



u 


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o 


CL. 


z 




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oi 


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GEOGRAPHIC DISTRIBUTION OF MULTIPLE SCLEROSIS 



133 



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be "o 

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134 



JOHN S. BARLOW 



world, but their trend with latitude can be compared, and hence the plots 
shown in Fig. 5 were constructed from the last column of Table IV. 

Although there is little to choose between the geographic and the geo- 
magnetic plots with respect to the better fit by the sigmoid curves that have 
been included, superimposition on the plots of Figs. 2, 3, and 4 is possible 
only if the geomagnetic parameter is chosen. 

From their data, Grashchenkov and his collaborators (1960) separately 
examined the effect of maritime climate on the frequency of multiple sclerosis 
by comparison of the statistics obtained for several maritime cities in the 



TABLE V 

Relative Hospital Admission Rates for Multiple Sclerosis 

IN Selected Maritime Cities in the Soviet Union * 







Geographic 


G 


eomagnetic 


Percentage 


of 


R 


dative number 


City 




latitude 




latitude 


cases 


with M.S. 




of cases ** 


Archangel 




65 




59 




3.2 






L06 


Riga 




57 




55 




2.8 






0.90 


Odessa 




47 




43 




L6 






0.53 


Sochi & Su 


khumi 43 




38 




0.58 






0.19 


Baku 




40 




34 




0.45 






0.15 



" Data from Grashchenkov et al. (1960). 

'' 1.0 corresponds to the mean of the percentages for the two cities of greater than 50° geomagnetic 
latitude (i.e., 3.0%). i 



1.2 - 



1.0- 



0.8 




0.0--- 
20 



y 



30 40 50 60 70 

GEOGRAPHIC LATITUDE 'N 




20 30 40 50 60 70 

GEOMAGNETIC LATITUDE 'N 



Fig. 6. Relative number of hospital admissions for multiple sclerosis in selected 
maritime cities of the Soviet Union (see Table V). 



GEOGRAPHIC DISTRIBUTION OF MULTIPLE SCLEROSIS 135 

Soviet Union. These data (Table V) also clearly indicate a greater fre- 
quency in the northern regions. Again, geomagnetic latitude is the better 
parameter if the results plotted in Fig. 6, are superimposed on those in Figs. 
2-4. 



Predicted Geographic Distribution of Multiple Sclerosis 

To permit comparison between the data represented in the preceding 
figures and those from future surveys or from other sources, geomagnetic 
latitudes corresponding to relative prevalences of 0.1 and 0.9 were deter- 
mined from the sigmoid curve in Fig. 2 and are approximately 38° and 48°, 
respectively. These geomagnetic coordinates are indicated as isoprevalence 
lines by solid lines in Fig. 7. For inhabited areas of geomagnetic latitude less 
than 38° (i.e., the area between the two 0.1 lines), relatively low prevalence 
ratios of the order of 4—6 per 100,000 population are predicted. North and 
south of the 0.9 lines in the northern and southern hemispheres, respectively, 
relatively high prevalence ratios of the order of 40-60 are predicted. A rapid 
increase of prevalence with increasing geomagnetic latitude is predicted to 
lie between the 0.1 and 0.9 lines in both hemispheres. 

The predictions from such a map appear to be in reasonably good agree- 
ment with much of the available data concerning the distribution of multiple 
sclerosis (Barlow, 1960). The general distribution being relatively common 
in northern Europe and in northern North America, but relatively uncom- 
mon in the Orient, South America, Africa, and the tropics appears to be in 
accord with the map. 



Comparison of Multiple Sclerosis with Hodgkin's Disease 

The geographic distribution of multiple sclerosis has been contrasted with 
that for Hodgkin's disease on several occasions (Ulett, 1948; Kurland, 1952; 
McAlpine et al., 1955), since Hodgkin's disease, at least in the United States 
and Canada, appears to show little geographic variation. It is of interest to 
compare statistics for the two diseases from the present standpoint. For this 
purpose, mortality statistics for the two diseases in several localities through- 
out the world, compiled by McAlpine et al. (1955), are compared 
in Table VI and are plotted in Fig. 8. The statistics have limitations and may 
be subject to considerable sampling error since only one year is represented 
for each locality. Nonetheless, it is apparent that the latitude trend of rela- 
tive mortality for multiple sclerosis is somewhat similar to that suggested by 
the prevalence data in Fig. 3. Moreover, there is possibly less scatter of the 
points about the sigmoid curve (reproduced from the geomagnetic plot of 
Fig. 2) for the geomagnetic plot than for the geographic plot. For Hodgkin's 



136 



JOHN S. BARLOW 




GEOGRAPHIC DISTRIBUTION OF MULTIPLE SCLEROSIS 



137 






Qi 



c^ 






QjJ~« 



O 






— 'O OO-^ OOO OOOO OO OO— •OO— '-^-^— ' 



CNJ O — ^ O — ' O O -^ OOOO -- -^' o o ^ O ^ -H -^ -^ ^i 



c^io_ o^— iin -^^^co o— '— 'in co-^ oindiooi — r^iOiD 

o" O OOO o o o o o o o" o o o o' o o o -^ — — < -^ 



mo i^coo iMcor^ ooco-H <oo^ -HC^IC0(yllnO'— 'f^co 

OO -hO-h OOO cJo'oo" OO 0-^-^o'oCMC<-)C^ioi 



Or^ COCTiO CO<T)tC OCTiCOCO CTltD r^c<0O'-<(>JO — <r>CO 
rn-t- -t--+'Tf co'-h-i- -f -V en 'T *+-+ cocnLO-f-t--t-in-t-Tf' 



C/D t/3 C/D C/D C/D C/2 c/l 



^=0 "S.^"^ ^,oco cnooo-, o' 



I— j-a u 
, «« 3 ,^ 

T3 N jO 



rt 



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rt 



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03 ?3 i-c 



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c - S g- 



138 




JOHN S. BARLOW 

MULTIPLE SCLEROSIS 



1.0 « I.ei DEATHS PER 100,000 POP. PER YEAR 




GEOGRAPHIC LATITUDE °N OR S 



20 30 40 50 

GEOMAGNETIC LATITUDE °N OR S 



HODGKIN'S DISEASE 



l.O = 1.45 DEATHS PER 100,000 POP. PER YEAR 




10 20 30 40 50 60 70 

CtOCRAPnC LATITUDE °N OR S 




K> 20 30 40 so 60 70 

CEOMACNETIC LATFTUDS °N OR ( 



Fig. 8. Latitude distribution of multiple sclerosis and Hodgkin's disease by relative 
mortality rates (see Table VI). 



GEOGRAPHIC DISTRIBUTION OF MULTIPLE SCLEROSIS 139 

disease, however, the latitude trend is considerably more poorly defined, 
although there is some suggestion of an increasing mortality with increasing 
latitude. Further, there is little to choose between the geographic and the 
geomagnetic plots for Hodgkin's disease. 

Latitude Effect for Cosmic Rays 

The statistics for multiple sclerosis that have been examined here, as well 
as those considered previously (Barlow, 1960), appear to suggest that the 
geographic distribution of this disease is better correlated with geomagnetic 
latitude than with geographic latitude. Since the phenomenon of cosmic 
radiation is the only one known to be related to geomagnetic latitude,^ it is 
appropriate to examine the latitude eflfect for various cosmic ray parameters 
for comparison with the latitude effect for multiple sclerosis. Variation with 
altitude must also be considered. For multiple sclerosis the available data 
do not indicate any clear variation with altitude (Barlow, 1960), whereas 
for cosmic rays the altitude effect is generally large compared to the latitude 
effect. Such is the case for the ionization produced by cosmic rays as deter- 
mined by an ionization chamber (Fig. 9). At sea level, the latitude effect 
between 0° and 50° is only 14%, a much smaller effect than is apparent in 
Figs. 2-6. Between 40° and 50°, it is even smaller. The altitude effect of 50°, 
however, is such that there is about 70% more ionization at an altitude of 
2,000 meters (6,500 feet) than at sea level. 

The latitude effect for multiple sclerosis thus is not in accord with that for 
the ionization produced by cosmic rays, except some of the data for multiple 
sclerosis are suggestive of a "knee" at about 50°, and a "knee" at this lati- 
tude is apparent in Fig. 9. Neither the meson component nor the nucleonic 
comp)onent (protons and neutrons) of cosmic rays near sea level had a 
latitude effect as pronounced as multiple sclerosis apparently has. 

More pronounced latitude effects are found at the top of the atmosphere 
(i.e. at about 80 kilometers or 50 miles), at heights where the atmosphere 
has not yet exerted its filtering and diffusing effects on the incoming cosmic 
ray flux. Thus, the total number of particles incident vertically at high lati- 
tudes is some ten times greater than the number incident at the geomagnetic 
equator (Fig. 10). Such a curve represents the flux for primary particles of 
all energies, and if specific energy ranges are examined, even more pro- 
nounced latitude effects are apparent, as indicated in the theoretically com- 
puted curves reproduced in Fig. 11. The sigmoid curve from Figs. 2-6 has 
been included in Fig. 11, and a close parallel is seen between this curve and 
that for primary cosmic ray protons of 4.5 Bev energy. This order of energy 

" The aurora borealis and australis appear to be indirectly related to cosmic radiation 
and the earth's magnetic field through the intermediary of the van Allen radiation 
behs (van Alien, 1959). 



140 



JOHN S. BARLOW 



7.5 
7.0 














-i~ 
































/ 










6.5 

6.0 

5.5 

5.0 

4.5 

4.Q 

2 3.5 
g 










Barometer 45cm ; 
Altitude. 4360m/ 


/ 
















« 


/ 




1.72 ions ' 
33 per cent 




D 2. 


-,tr^ 


_fl__ 








_«__ 


- 




























































Barom 


eter € 


Ocm 


o 

-J3W 










r 3.0 

c 

^ 2.5 
2.0 


A TITUQc c.\jyj\ji\\ 










U D 




A 


n^ 


^ 8 


.56 
22p 


ons 
er ce 


nt 








■) 


cm - 














^^ 


\fK 


, 








n r^m 




hr— "2 


, 


.22 ions 




1.5 
1.0 
0.5 














I J\ 


, 














^Je^ L 











































10 20 30 40 50 60 70 80 90 
Geomagnetic latitude 

Fig. 9. The latitude effect for the ionization produced by cosmic rays at different 
altitudes. The altitudes 2,000 and 4,360 meters correspond to 6,500 and 14,000 feet, 
respectively. (From Janossy, 1950, and adapted from Compton, 1933.) 



is somewhat more than twice the energy that corresponds to the rest mass 
of the proton or neutron and hence is in the range of the threshold energy 
for production of nucleon-antinucleon pairs (Segre, 1958). It is also in the 
same energy range as that required for a primary proton to penetrate the 
atmosphere and come to the end of its path in a thickness of some 10-60 cm 
of water equivalent (Aron et al, 1949), i.e., to come to rest in the brain or 
spinal cord of a human in the open air. The greatest biologic effect of 



GEOGRAPHIC DISTRIBUTION OF MULTIPLE SCLEROSIS 



141 



0.8 



0.4 




20 30 40 

Geomagnetic latitude 



Fig. 10. Vertical flux of cosmic ray particles near the top of the atmosphere. (After 
Curtis, 1956, and from Puppi and Dallaporta, 1952.) 



e48.3 

100^==* 




Fig. 11. Dependence of primary cosmic ray intensity on magnetic latitude (\). 
The ordinate represents the relative intensity for each of the various energies {p = 
energy of protons in billions of electron-volts; other parameters shown on the cur\'es 
are not relevant to the present discussion), expressed as the per cent of the maximum 
possible intensity for that particular energy. (From Richtmyer et al., 1955, and adapted 
from Lemaitre and Vallarta. 1933.) 



protons appears just before the end of their trajectory (Malis et al., 1957). 
However, the number of protons of a similar terminal energy at the surface 
of the earth which have been produced by the complex interaction of 
primary cosmic rays with the atmosphere is far greater than the number of 



142 JOHN S. BARLOW 

primary protons in this category (Wilson and Wouthuysen, 1958), and the 
latitude effect for such secondary protons is much less than that for primary 
protons (their altitude effect is also appreciable). For these reasons, the 
striking similarity between the two curves in Fig. 11 must be considered 
fortuitous. 

Corrections to the Geomagnetic EflFect 

Since a rather narrow range of geomagnetic latitudes appears to corre- 
spond to the zone of rapid increase in prevalence of multiple sclerosis, 
further clarification of the details of the exact relationship between the lati- 
tude effect for cosmic rays and the earth's magnetic field possibly may be of 
importance for the present study (Katz et al., 1958). Thus, corrections for 
local variations in the earth's magnetic field may be necessary to provide 
a better fit for observed cosmic ray phenomena than geomagnetic latitude 
per se. The dashed lines in Fig. 7 indicate isoprevalence lines constructed on 
this basis and determined from data for cosmic rays published by Quenby 
and Webber (1959). There is little difference in the location of the two sets 
of isoprevalence lines in inhabited areas of the world except in central Asia, 
and the circles in Fig. 5 indicate the corrections that would occur on this 
basis for the two locations in this area represented in the figure. 

Discussion 

Cosmic radiation has been implicated as a factor in human disease in 
other studies (Morris and Nickerson, 1948; Wesley, 1960), but the marked 
latitude effect for multiple sclerosis appears to provide a greater possibility 
for distinguishing between geographic and geomagnetic latitude as a param- 
eter, a test which originally firmly established the relationship between 
cosmic rays and the earth's magnetic field (Compton, 1933). 

The present data and that examined previously (Barlow, 1960) appear to 
suggest that cosmic radiation in some way may be related to the occurrence 
of multiple sclerosis. 

Since at a given location multiple sclerosis apparently is distributed ran- 
domly among the susceptible population (Kurland et al., 1955) and since 
plaques of demyelination of this disease are largely randomly distributed 
in the white matter (Adams and Kubik, 1952; McAlpine et al., 1955), it 
would be an attractive possibility to attempt to relate a randomly occurring 
cosmic ray event to the trigger mechanism that Lumsden (1951) suggests 
may occur in the initiation of plaques of demyelination. Several considera- 
tions militate against such a direct relationship, if there is any relationship 
at all between cosmic radiation and multiple sclerosis. Among these consid- 



GEOGRAPHIC DISTRIBUTION OF MULTIPLE SCLEROSIS 143 

erations are the generally greater level of radiation from terrestrial sources 
of naturally occurring radioactivity- as compared with that from cosmic 
radiation (Libby, 1955; Neher, 1957; Solon et al., 1960) and the lack of 
an altitude effect for multiple sclerosis. 

Since direct effects of cosmic rays at the earth's surface cannot be impli- 
cated, it is of interest to examine the latitude distribution of atomic nuclei 
made radioactive by cosmic ray events. These radionuclides include T (the 
hydrogen isotope, tritium), Be^ Be^", C\ Na^^ P^-, P^^ S^^ and CF^ (Suess, 
1958). The production rate for cosmic ray-induced radionuclides is said to 
be greater by a factor of four at the poles than at the geomagnetic equator, 
following the latitude effect for the neutron component of cosmic rays (Suess, 
1958; Kaufman and Libby, 1954; Simpson, 1951). Their concentration at 
the surface of the earth, however, is dependent on several additional factors, 
including half-life, diffusion in the atmosphere and the oceans, and meteoro- 
logic factors. Thus, the half-life of C^* (5,570 years) is so long that diffusion 
processes tend to minimize any variation of its concentration with latitude. 
Diffusion effects should be relatively small for elements with short half-lives, 
P^- for example (14.5 days). 

In any event, it would not appear possible that the latitude distribution of 
any cosmic ray-induced radionuclide would be as pronounced as that for 
multiple sclerosis. A latitude effect for the distribution of fall-out from heavy 
cosmic ray primary nuclei might be considered additionally (Wesley, 1960). 

Consideration of the problem of the latitude distribution of radioactive 
nuclei associated with cosmic rays is even further complicated by the fact 
that some of these nuclei are produced in the explosion of atomic and hydro- 
gen bombs. Thus, the amount of T that has been produced by hydrogen 
bombs is comparable to or larger than the total inventory of natural T on 
the surface of the earth, and artificially produced C^* had by 1957 increased 
the C^* content of atmospheric CO2 by about 10% (Suess, 1958). Although 
there is a pronounced distribution with latitude for fall-out debris, at least 
as indicated by Sr^" in the northern hemisphere (Fig. 12), the variation is 
with geographic latitude and not with geomagnetic latitude. It should of 
course be remembered that multiple sclerosis was well-known as a clinical 
entity long before the era of bomb testing. 

Linear vs. Nonlinear Dose-Response Relationships 

The above considerations concerning comparisons of the geomagnetic 
latitude distribution of multiple sclerosis with those for various cosmic ray 

^Gentry et al. (1959) have recently found that areas with increased rates of con- 
genital malformations in New York State appear to be associated with increased levels 
of background terrestrial radiation. 



144 



JOHN S. BARLOW 



1.0 - 



t^ 0.8 - 




20 30 40 50 60 

GEOGRAPHIC LATITUDE 



1.0 - 



?: 0.8 - 



o'-r 




30 40 50 60 

GEOGRAPHIC LATITUDE 



Fig. 12. Relative concentration of Strontium-90 versus geographical latitude. 

a. In the stratosphere, averaged over the period November 1957-November 1958. 
1.0 = 12 mc per square mile. (Data from Feely, 1960). 

b. On the ground in November, 1958. 1.0 = 4.1 mc per square mile. (Redrawm 
from Wexler, 1960.) 



parameters are based on the assumption of a one-to-one {i.e., linear) corre- 
spondence between the exposure and the number of cases of the disease. 
Should a nonlinear relationship obtain, it theoretically would be possible 
for a more pronounced latitude distribution for the disease to result from a 
cosmic ray phenomenon of a given latitude distribution. (The altitude effect 



GEOGRAPHIC DISTRIBUTION OF MULTIPLE SCLEROSIS 145 

for such a phenomenon would still have to be small compared with its 
latitude effect.) Such a nonlinear relationship between inciting agent and 
disease can obtain, for example, if the susceptibility of individuals in the 
population is distributed in a normal (Gaussian) manner. A normal distribu- 
tion for susceptibility and the concomitant nonlinear relationship between 
per cent incidence and dose rate at low doses appears to obtain for the 
occurrence of tumors in mice following exposure to ultraviolet light, and it 
has been suggested that a similar relationship may occur for the appearance 
of cancer in man following exposure to ionizing radiation (Blum, 1959a,b). 

Duration of Exposure to Inciting Agent 

Whatever is the cause of multiple sclerosis, a most interesting question 
arises in connection with the incubation period for the disease. Acheson et 
al. (1960) have pointed out that if the inciting or protective agents are 
prolonged in their effect, then the important factor in the history of indi- 
vidual cases will be that of place of residence over an extended period; 
alternatively, only the place of residence early in life might be the important 
factor. These workers suggest that a long incubation period may be impli- 
cated by their own findings as well as by the observation of Dean (1949) 
that multiple sclerosis is almost unknown in persons of European stock born 
and raised in the Union of South Africa, whereas it is more frequently 
described in persons born in Europe who have emigrated to South Africa. 
An analogous observation in Israel by Rozansky (1952) is being verified 
by Alter (1960). 

The present approach does not help in elucidation of this question, for 
it is conceivable that radiation could have either a single-shot effect, perhaps 
analogous in animals to the graying of hair produced by heavy cosmic ray 
nuclei at very high altitudes (Chase and Post, 1956), or alternatively it 
might have a cumulative effect, as does the carcinogenic property of ultra- 
violet radiation. 

Possible Experimental Approaches 

Despite the difficulties of establishing more than a correlative (and there- 
fore possibly fortuitous) relationship between cosmic radiation and multiple 
sclerosis, it is perhaps in order to consider briefly some possible experimental 
approaches to the problem, particularly if additional carefully collected epi- 
demiologic data substantiate the results so far obtained. These experimental 
approaches difTer somewhat according to whether it is the place of residence 
in adult life, or in early life, that is established as being the important factor 
in the geography of the disease. 



146 JOHN S. BARLOW 

The early plaques of demyelination in multiple sclerosis appear pre- 
ponderantly around small veins (Adams and Kubik, 1952), and a similar 
localization appears for the demyelinative lesions of experimental allergic 
encephalomyelitis in some animals (Mc Alpine et al., 1955; Waksman, 1960). 
Further, induction of the lesions of the latter disease in predetermined loca- 
tions in the brain by use of physical agents has been reported by Clark and 
Bogdanove (1955). It might be of interest to determine whether lesions of 
experimental allergic encephalomyelitis could also be induced in predeter- 
mined locations by low doses of radiation from well focused beams of high 
energy particles (for example, of appropriately filtered high energy protons). 
Perivenous staining with trypan blue has been rep>orted in the demyelinative 
plaques of multiple sclerosis (Broman, 1949) and in lesions of experimental 
allergic encephalomyelitis in animals (Barlow, 1956; Waksman, 1960) ; a 
similar focal staining might be looked for following focused irradiation at 
low doses, since it is a known finding with much larger doses (Clemente and 
Hoist, 1954) . Should positive results be found from either of these experi- 
ments, a possible protective eflfort might be explored for some of the agents 
(e.g., cysteine) which are known to lessen the biological effects of ionizing 
radiation. 

If place of residence early in life is the imp>ortant factor, then possible 
relationships (direct or indirect) might be explored between radiation and 
immunochemical processes in early life which might underlie demyelinative 
processes in adult life. 

In connection with the experiments outlined, it should be noted that if 
cosmic radiation is at all related to multiple sclerosis, the mechanism of its 
action is likely to be different from the biologic effects of cosmic rays at high 
altitudes (Yagoda, 1957; Schaefer, 1958). 

Finally, the possibility should be kept in mind that some other factors 
(for example, certain types of infections, perhaps not known) might act as 
intermediaries between cosmic rays and multiple sclerosis. 

Summary and Conclusions 

Multiple sclerosis appears to exhibit a fairly well marked geographic dis- 
tribution, being appreciably more frequent in northern than in southern 
latitudes in certain areas of the world. It is generally agreed that this geo- 
graphic distribution must be taken into account in any satisfactory theory 
of etiology of the disease. Possible correlates of the geographic distribution 
which previously have been advanced have not appeared entirely satisfactory; 
certainly geographic latitude itself appears to be a poor correlate. Several 
independent sets of statistics on the distribution of the disease were examined 
from the standpoint of geomagnetic latitude, a parameter that is related to 



GEOGRAPHIC DISTRIBUTION OF MULTIPLE SCLEROSIS 147 

the earth's magnetic axis in the same way that geographic latitude is related 
to the earth's axis of rotation. That the distribution of multiple sclerosis 
might be examined in this way was suggested by the fact that a variation 
with geomagnetic latitude is well known for the intensity of cosmic rays. 
The frequencies of the disease in widely separated areas were found to vary 
in a systematic manner with geomagnetic latitude; therefore, the possibility 
arises that cosmic rays in some way might be a factor in the occurrence of 
the disease. The latitude distribution for the disease bears some resemblance 
to that for a particular component of the primary cosmic radiation at the 
top of the atmophere; no similarly good correlate is apparent, however, 
among the various cosmic ray components at the surface of the earth, hence 
the similarity may be entirely fortuitous. Some possible intermediary factors 
examined appear to offer no great promise. A world map, on which is indi- 
cated the expected geographic distribution of multiple sclerosis on the basis 
of geomagnetic latitude, is presented for comparison with results of future 
surveys on the disease so that the present results may be evaluated further. 

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150 JOHN S. BARLOW 

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GENERAL DISCUSSION 

Paul Henshaw (U. S. Atomic Energy Commission, IVashingtoti, D. C): I am 
indeed interested in the information Dr. Rugh presented with his spectacular slides 
of early embryos. Seeing evidence of deteriorating embryos at an early stage is to 
be expected in view of what is known about the quality of germ cells and the 
uterine bed in some situations. Certainly, degeneration is inevitable as a consequence 
of some of these conditions. Dr. Rugh has called attention to the kinds of ab- 
normalities that occur following different levels of exposure to germ cells and to 
early embryos, and it was particularly interesting that abnormalities show in 
organisms exposed to doses of 5-15 r. This is an extremely low level of irradiation, 
and I am sure it will be quoted repeatedly. I feel, therefore, that we should ask 
questions about the confidence he has in the findings. Dr. Rugh has shown abnor- 
malizes which indeed do show in samples of organisms that have been exposed 
to low doses of radiation, but such abnormalities will show as a consequence of 
other agents as well. I would be pleased if Dr. Rugh would cite the specific 
evidence he has that permits him to say the low level changes are due to radiation. 
The second point pertains to the abnormality that showed in the third generation. 
Were these actually due to radiation change involved in a germ cell? We know 
something about how cerebral hernias will result from damage to neural fold 
primordia, the failure to close the neural crest, which permits the brain to turn 
inside out. This is a developmental abnormality not connected with germ cell 
damage. If the same can come from irradiation of germplasm, this is exceedingly 
interesting. I would like Dr. Rugh to indicate whether he feels there is evidence 
that cerebral hernias may result from mutations in germ cells. Third, Dr. Rugh 
has emphasized that low levels of radiation produce developmental abnormalities 
and went so far as to call attention to the possibility that a large portion of the 
naturally occurring developmental abnormalities may be due to background radia- 
tion. If I were a physician, I think I would go along with Dr. Rugh's warning and 
be cautious about any exposure of embryos or germ cells, but I am a laboratory 
man. I would like Dr. Rugh to give his strongest evidence that environmental 
radiation is having a significant effect. Does he feel that background radiation 
can or does account for a substantial proportion of the abnormalities, having 
knowledge as we do that many things can produce the kind of abnormalities des- 
cribed? 

Roberts Rugh (Columbia University): In regard to your first question con- 
cerning whether we are dealing with low level effects of radiation or possibly 
other traumatic effects: We are currently studying the embryos found in 98 pairs 
of uteri of mice exposed to 15 r at 1/2 days, because we felt this sort of statement 
had to be quantitative and proven. We will have statistical data from over 1,000 
embryos which received 15 r at 1/2 days with an appropriate number of controls. 
It is true that we get anomalies without irradiation, that is, without superimposed 

151 



152 GENERAL DISCUSSION 

irradiation from laboratory sources in addition to background irradiation; and it is 
also true, as you saw from the first slide, that we get about 7% death and resorp- 
tion of embroys in the controls. This may be due to cosmic radiation or natural 
background radiation, or even may be due to genetic causes. We do not know. 
We have been dealing with exencephaly as an anomaly that was definitely pro- 
duced by irradiation. We have never seen this anomaly except following irradiation, 
and we have produced this at the low level of 15 r in the early embryo. The second 
question dealt with genetic effects. I did not state that the three generations of 
exencephaly found following irradiation of the ovary or the testes were due to 15 r. 
It was due to a much larger dose as for instance, 100 r to the ovary of the great- 
grandmother. The important point was that the effect was carried through three 
generations. Obviously, it would be necessary to determine the statistical frequency. 
However, that it occurs at all following irradiation of the ovary is of concern to 
every potential mother. The point to emphasize is that this anomaly was produced 
by irradiation of the germ cells and was found in three successive generations. 
It therefore had a genetic origin. Snell showed about 1935 that x-irradiating the 
testes and having the male mate with a normal female produced in the second 
generation something like 35% of such anomalies. We have carried it from both 
the sperm and the egg through several generations, and we were simply empha- 
sizing that this anomaly appears to be similar, whether it is derived from an effect 
on the chromosome or an effect directly on neurogenesis. How it is produced 
genetically we do not know, but having had considerable experience in experimental 
embryology with amphibia and chicks, it seems to me it is probable that the 
damage was to chromosomes which at the time of gastrulation caused such dis- 
ruption in organogenesis that any gross change could follow. This happens to be 
one that is simply produced due to failure of closure of the cranial roof and loss 
of neuroblasts and probably osteoblasts during development. The third question 
related to the dangers of cosmic radiation. Like taxes, we are all faced with 
cosmic and natural radiation, and there is nothing much we can do about it. This 
may actually be good for evaluation! I think, however, in line with the last paper 
and those of Drs. Gentry, Wesley, and others, it may be proven statistically that 
there is some correlation between the amount of background radiation and the 
incidence of congenital anomaHes. If our thesis is correct, at this early stage of 
development the embryo is so extremely radiosensitive that 5 r causes a 10% 
increase of resorptions and 15 r causes exencephaly in the embryos which develop 
later. 

Percfval Bailey (University of Illinois): This afternoon I am handicapped by 
lack of intimate knowledge of the embryonic cerebral cortex and by the quantity 
of the material presented. You cannot really judge histologic material by a few 
projections. The material presented here seemed inadequate for any fine cytologic 
study. I suppose I should be happy that it is so, because that leaves an oppor- 
tunity for somebody else to make a good cytologic study of the effects of radiation 
on the cells of the cerebral cortex, with more adequate cytologic technic. 

Orville Bailey (University of Illinois): Dr Brownson, were you radiating the 
whole animal or the head only? And did you conclude that fractional doses of 
radiation produced less or more effect than the same total dose at one time? How 
long did the animals live? 



GENERAL DISCUSSION 133 

Robert H. Brownson (Medical College of Virginia): To answer the first 
question, this was total head x-ray. Second, this was a cumulative exposure, and 
we have not compared the total radiation effect accumulated at one time with it. 
Concerning the effects of radiation, the alteration was cumulative in the direction 
of change which was more quantitative than qualitative. The effects we saw did 
not seem to show more severity in themselves individually, but more intensity 
through the actual quantity of such change. The total picture which we saw at the 
end, beginning with 2,000 r as a minimum dose, demonstrated individual changes 
at 228 days similar to those changes that we could see using the 5,000 r level 
following a shorter postirradiation. The problem was to have these animals 
survive. Many did not survive 228 days, probably due to being stressed with an 
additional nutritional deficiency. In testing these animals psychologically, we had 
to deprive them of some food, and this influenced the death rate which accelerated 
with the increasing cumulative radiation. The group which exhibited the greatest 
change was the 5,000 r cumulative group, which were not subject to any type of 
psychologic testing and went through a relatively normal span of 228 days. Much 
of the probelm in correlating the changes of one of the animals with 18-20 r with 
5,000 r was that these animals had gone through 228 days normally while the 
5,000 r animals did not survive. Most of the changes were quantitative and in 
general increased in direct proportion to cumulative dosage and, to a degree, to 
time after exposure. 

Orville Bailey: In the terms I ordinarily use it seems as the intensity of the 
radiation goes down, the amount of damage per total dose also falls. One can 
build up almost grotesque amounts of x-ray dosage without damage if given slowly 
enough over a long period of time. Most of the lantern slides which Dr. Brownson 
showed were in the acute phase of the reaction which is difficult to evaluate. The 
focal neuronal changes that were described seemed like small foci of "dark 
neurons," the change which Dr. Gammermeyer has studied. They are artifact 
or at least, reflect some terminal state of activity in that particular cell. Most of 
the changes in the Purkinje cells, as Dr. Vogel and associates have demonstrated, 
are quite frequently found in control monkeys. 

E. C. Alvord (University of Washington School of Medicine): I would like 
to stress one minor theme that was developed by Dr. Rugh and has recurred in 
rather low notes through most of these papers. This is the concept that the body 
as a whole is made up of a mosaic of many structures, each of which has vastly 
different sensitivities to radiation. This concept of a mosaic also applies within 
a part of the body, namely the nervous system itself. There are a number of 
syndromes that have been delineated, particularly by Maisin of Belgium, on the 
basis of survival times following various doses of x-rays to various parts of the 
body. He speaks of a "delayed head syndrome," which occurs in rats after 1,000 r 
to the head, the rats dying about 5 months later, and of an "oropharyngeal 
syndrome," which suddenly appears at 1,500 r and cuts the survival time down to 
10 days. I would like to ask Dr. Brownson to define the exact site of the irradia- 
tion to the heads of his rats. I doubt that this included the whole head, since 
Maisin and others have found it difficult for rats to live beyond 10 days following 
irradiation of the whole head with 1,500 r or more. This "oropharyngeal syndrome" 
has been found to be due to the inclusion of the oral pharynx, tongue, and lower 



154 GENERAL DISCUSSION 

jaw of the animal, damage to these particularly sensitive areas causing death by 
means that are not clearly defined. It would be particularly interesting if repeated 
doses of 1,000 r can avoid this syndrome and allow as much as 5,000 r to be given, 
but I would suggest that Dr. Brownson has irradiated only the forebrain so that he 
sees relatively little of the change in the cerebellum because of a slight rostral 
advancement of the posterior margin of his x-irradiation. My own experience is 
only with the adult animal. I am sure that in the adult guinea pig one has to 
include the cerebellum and has to go well below the cerebellum to produce 
neurologic signs and death of the guinea pig in a short time. This leads me to 
Dr. Schjeide's paper, in which, unfortunately, the wet weights are not available. 
I would predict that, when the wet weights become available, the most striking 
chemical change will be in the degree of hydration with marked edema of the 
cerebellum. Dr. Sauer, have you with tritiated thymidine been able to apply this 
at certain times after the irradiation, with the idea of establishing whether these 
DNA bodies are dead or still metabolically active? 

Wolfgang Zeman (Indiana University Medical School): I think we should 
strive for a more accurate definition of cumulative or fractionated doses. In 1949, 
I tried to arrive at an understanding as to the radiobiologic effectiveness of frac- 
tionated doses as compared to a single dose. My data at that time were rather 
scanty, but in the meantime Lindgren (Stockholm) arrived at a simple formula 
for converting fractionated doses into single exposures. He determined the x-ray 
dose which was necessary to produce radiation-induced brain damage in 50% 
of rabbits. He used various single and cumulative dosages and found that in plot- 
ting the morbidity dose (50%) in r logarithmically against the total amount of 
days over which this dose was given, also logarithmically, a straight line results 
which has a slope of about 0.26 for the adult rabbit brain. In other words, a dose 
of say 2,000 r given in one day, compares to a dose of 2,000 r times lO^-^'^ given 
over 10 days. For the human brain the slope has been shown to be about 0.34, and 
it stands to reason that each different species does have a specific slope. I would 
predict that within one species, the slope might be dependent on the develop- 
mental stage. I wonder whether Dr. Brownson would be good enough to convert 
his data into terms which would make for an easy comparison to the radiobiologic 
effectiveness of cumulative doses with single dose exposures. 

L. J. Peacock (University of Georgia): I would like to ask Dr. Brownson 
whether the decline in response rate in his rats was due to an error in their timing 
behavior or to a decrease in motivation. That is, was there a decline in the over- 
all food intake of these animals, or was it a matter of their not being able to 
properly time the intervals and schedule? 

Robert H. Brownson: Dr. Alvord, the radiation instrumentation was conducted 
by our physicists in the biophysics department. Each animal was prepared by 
placing it in a cage in which the head was elevated out of the cage and held by 
a clamp with the remainder of the animal shielded. A Victoreen Chamber R-Meter 
was used to monitor the dosage. Each animal received 1,000 r delivered at the 
rate of 237 r per minute. The animal was given 1,000 r per week, so that it 
accumulated as scheduled per week the desired total roentgen dosage. Our ex- 
perience with guinea pigs has indicated that they are more liable to radiation 



GENERAL DISCUSSION 155 

death than rats. Our rats did well with 5,000 r cumulative total head exposure at 
the end of 228 days, provided they were not stressed as they were when they were 
placed in the Skinner box and tested for positive food reward. To reply to Dr. 
Peacock's question: to our way of thinking this change in the animal's behavior 
as related to its performance in the Skinner box was one that seemed to be motiva- 
tion. We tested our animals between and after each dosage and following the end 
of the first 5 weeks on food intake. The food intake of the x-irradiated animals 
versus the controls was not so different as to make us think that this was the whole 
picture. The controls when placed on a deprivation diet maintained their normal 
weight, which tended to climb slowly. We believe there is a definite food problem 
involved, but there may have been one of motivation also. We are now utilizing 
shock avoidance and positive food reward together in anticipation that this will 
help clarify the matter. 

John L. Falk (Harvard School of Public Health, Boston, Massachusetts): 
I was happy to see that Dr. Brownson was using long-term testing. Short-term tests 
involving food intake or various food-motivated performances might cause radia- 
tion sickness. Did Dr. Brownson use a variable interval schedule? We have been 
making readings in medial nuclei of the hypothalamus and getting increases in 
bar-pressing rates on variable interval schedules. Since there did seem to be some 
hypothalamic involvement in Dr. Brownson 's animals, I wonder if perhaps there 
was more involvement in the lateral nuclei, possibly indicating classical aphagia? 

Robert H. Brownson : I think the only answer we will have to this question 
is dependent on the results obtained with shock avoidance. We anticipate improved 
testing methods in our future plans. The schedule was aperiodic, and the tapes 
were run for 45-minute intervals. The periods ran 4-224 seconds apart with an 
average of 62 seconds between reinforcements. 

Cornelius A. Tobias (University of California, Berkeley, California): I had the 
pleasure of discussing with Dr. Barlow his interesting statistical findings and trying 
to encourage him and other people who are taking a similar approach to the 
explanation of some diseases similar to this as to etiology. However, there are 
several aspects that need further study and improvement. It seems to me that if 
multiple sclerosis is due to radiation, it is most likely that it should be due to early 
effects in the embryologic or even germ cell stage. It appears that neither the 
studies by Dr. Hicks nor Dr. Rugh show that multiple sclerosis is a frequent 
occurrence in animals developing from irradiated embryos or germ cells. Secondly, 
findings such as these should be correlated with other findings which were also 
discussed in Dr. Rugh's paper. For example, those by Gentry, who finds that in 
certain areas of the United States with high natural radioactivity background 
congenital malformations occur more frequently than elsewhere. If the Barlow 
and Gentry studies do not correlate, then it would seem that it is not the low 
ionizing component of cosmic rays that would cause the effect in multiple sclerosis, 
but some other component of cosmic radiation that occurs perhaps only rarely. 
There are some cosmic ray phenomena for which further work may be needed 
before correlations can be established. For example, the so-called Auger showers 
which are energetic showers arrive at ground level from primary cosmic rays which 
can give well measurable doses to a single individual. On the average one such 



156 GENERAL DISCUSSION 

shower passes through the body of an individual once a year. Another possibility 
is that a small percentage of the primaries that come in would come down to 
ground level and would produce highly ionizing secondaries. If highly ionizing 
particles are more eflFective than x-rays, one could perhaps understand the lack of 
results in x-irradiated animals. One would still expect to find an altitude effect; 
for example, multiple sclerosis incidence in Denver should be higher than in New 
York. Secondly, one would expect that in some way the incidence of multiple 
sclerosis would correlate with the 11-year cycle. The primary cosmic radiation 
events near the North Pole change radically in this 11 -year sunspot variation 
period, and perhaps babies bom in periods of low sunspot activity might exhibit 
a higher statistical incidence of multiple sclerosis. If heavy particles should cause 
multiple sclerosis, this hypothesis could be tested by exposing embryos to radiations 
produced in accelerators, such as the heavy alpha particles and other nuclei. 

John S. Barlow (Massachusetts General Hospital): The points made by Dr. 
Tobias are well taken; I might make a few additional comments. Multiple sclerosis 
per se is known only in man. There are naturally occurring demyelinating diseases 
in animals, but as far as I know these exhibit no latitude effect. At a given latitude, 
there are regional variations in the occurrence of the diseases, and it is an inter- 
esting suggestion to determine whether these are correlated with variations in 
background terrestrial radiation. The Auger showers may well be of biologic 
importance, but I think that the energy of the original cosmic ray particles giving 
rise to such showers is so great that no latitude variation for the showers would 
be expected. Those few primaries that do reach the earth's surface certainly would 
have a high relative biologic effectiveness, but they apparently are far over- 
shadowed in numbers by similar secondary particles for which the latitude effect 
is not very pronounced between 40° and 50° geomagnetic latitude and for which 
an appreciable altitude effect is present. The question of variations with the 11-year 
solar cycle merits further investigation. For several of the recent surveys of mul- 
tiple sclerosis, data for a 10-year period were collected, and no systematic fluctua- 
tion with time is apparent from these data. They have not, however, been exam- 
ined, as far as I know, from the standpoint of year of birth of the patients, as 
suggested by Dr. Tobias. Dr. John A. Simpson, of the University of Chicago, has 
informed me that the solar variation at 50° geomagnetic north is about 25% and 
decreases to some 6% at the geomagnetic equator; an effect of this size might 
well be obscured in statistics for the disease collected for localities of latitudes of 
50° or less. Studies in localities further north would be of particular interest from 
the standpoint of the solar cycle. 



SUMMATION BY CHAIRMAN* 

Samuel P. Hicks 

Harvard Medical School and New England Deaconess Hospital, Boston, Massachusetts 
The investigators have given summaries, and the discussants have added to this 
invaluably by emphasizing important points and questioning others. I will comment 
on matters that were not discussed and add information about the mechanisms of 
retinal, cortical, and cerebellar malformations produced in fetal and neonatal 
rats by 150-300 r of 250 kv conventional x-rays, a dose range widely used because 
of its selective effect and tolerability. The malformations are of prime importance 
to many of the experimental and behavioral studies to be reported later. 

In presenting his experiments Dr. Rugh has raised the important question of 
whether low doses (a few r) may impair the development of the embryo long 
before it begins to differentiate a nervous system. It is well-known that such early 
embryos have extraordinary powers of regulation and restitution, and a substantial 
number of cells may be lost; yet apparently normal individuals result. Dividing 
early embryos into two parts may result in two apparently norma! individuals. 
What we don't know is whether such embr)'os are really normal, although they 
have been reported to be so. The work Dr. Rugh and others are doing aims at 
exploring one aspect of this. At the morphologic level, the problem remains one 
of establishing a statistically sound relationship between the presence of necrotic 
cells in early embr^^os and an effect of low doses of radiation. There is no problem 
with higher doses — cells are killed. "Spontaneous" cell death has a way of turning 
up in a variety of circumstances during development, sometimes as a necessary 
normal process. As the dose of conventional x-rays is increased above 20 r at 
almost any stage of embryonic life, the number of dead cells increase, yet it is 
difficult to show that development has been impaired. In some later stages, for 
example when the neural folds are forming, the rat embr>'o can recover to a 
remarkable degree from excessive cell loss after 100 r or even 200 r, and it does 
so in a manner such as that described long ago in amphibians by Harrison and by 
Detweiler. Cpnira.ry to \:t:hatjp.r^ Rugh said, injuredpartsdo catch up successfully. 
In the face of this remarkable capacity for ernbryos toregulalestnicture after 
cell loss, we may have to look at other parameters when the resulting animal 
looks normal, but does not measure up in some aspects of function or behavior. 
Mutations and chromosome aberrations in the early embryonic somatic cells, 
alterations in other organ systems that indirectly affect the brain, and the indirect 
effects that irradiation of the mother has on her fetuses are some things to be taken 
into account. 

Dr. Brizzee's approach to the study of the effects of radiation on cortical growth 
is a new and promising one because some of the divided doses he gives probably 
kill few cells. Sixty r will kill some of the primitive cells. The single doses around 

* This research was supported by the A. E.G., the U.S. Public Health Ser\'ice, and 
the United Cerebral Palsy Association. 

157 



158 SUMMATION BY CHAIRMAN 

200 r employed by us and others extirpate certain classes of primitive proliferative 
and migratory cells and at a given stage produce specific deficits which are 
translated by ensuing morphogenetic sequences into adult patterns of cortical and 
other malformations. Dr. Brizzee's effects seem to be different and involve sub- 
lethal alterations of the differentiating neurons and glia. He reports that the over 
all cytoarchitectural pattern in the cortex is well organized in rats treated, say, 
with 25 r successively on fetal days 10 to 17, yet there are significant cytologic 
deviations from normal. These include alterations of the neuron, of the size 
of the nucleus in relation to cytoplasm, and in how closely packed the cells are. 
Some neurons were much too large. Closer packing of cells may have reflected 
subnormal proliferation of dendrites or other deficiencies in fiber development. 
What controls the size of a cortical neuron? How much influence do afferent 
fibers have on determining cortical cell types, and what alterations in DNA and 
RNA might lead to these cytologic abnormalities? Dr. Brizzee's further studies 
on the morphogenetic sequences of events may tell. 

Dr. Sauer, extending the studies of the late Prof. F. S. Sauer on the nature of 
the proliferative neuroepithelium, has come up with a new concept of partially 
destructive injury to the nucleus of the radiosensitive primitive neural cells. We 
had always thought that the quickly destructive effect of 200 r on the postmitotic 
migratory and other sensitive cells was an all-or-none phenomenon. Histologic 
studies of a series of embryos removed by Cesarean section at hourly or 2 hour 
intervals up to 9 hours after exposure still confirms this for the most part, but 
there is probably no discrepancy in our findings. In the chick, 200 r kills relatively 
fewer cells than in the rat, and Dr. Sauer clearly showed that after this lesser 
injury sublethal effects occurred. What happens to these partially incapacitated 
cells? Do they grow up to be abnormal neurons like those in Dr. Brizzee's rats? 
Some of his doses in rats may have produced effects corresponding to those 
following 200 r in the chick. 

The malforming effect of radiation on the developing mammalian central 
nervous system and retina depends on factors that include the stage of develop- 
ment, the individual growth characteristics of the species, and the doses of radia- 
tion. The dose largely determines what cells are killed and, therefore, what kind of 
malformative sequences of growth will be set in motion. Considerable data are 
now at hand on the morphology, the mechanisms, and the reproducibility of the 
malformations induced in albino rats by 200 r of conventional 250 kv x-rays when 
given at any stage from the 9th day of embryonic life to more than a week after 
birth. The acute extirpative effect of this exposure seems to be chiefly limited to 
the post-mitotic and primitive migratory neural cells, but obviously some chromo- 
somal damage with delayed cell death must also occur. Figure 1 indicates in 
schematic form how different doses kill cells in the young brain. The cerebral 
vesicle of a 17 day fetal rat is represented and shows that the neuroepithelial zone 
is a thick pseudostratified layer of tadpole-shaped cells which replicate their 
chromosomes in the outer part of this zone and slide in to mitose in the lining. 
This was demonstrated by F. C. Sauer in 1935 and confirmed by Watterson et al. 
(1956), M. E. Sauer and Chittenden (1959), Sidman et al. (1959; Sidman, 1961), 
and by Hicks et al. (1961a, b). Postmitotic cells are shaded, and they are the 



SUMMATION BY CHAIRMAN 



159 




DEVELOPING CEREBRAL WALL 
INJ THE RAT 



6^& 



20 DAYS 



Fig. 1. Schematic representation of the neuroepithelium. (Adapted from Hicks 
and D'Amato, 1960a.) 



principal radiosensitive ones, we believe. By radiosensitive we mean killed and 
visibly necrotic in 2 or 8 hours. The threshold of this effect is about 20-30 r. 
Above 200 r the selectivity is gradually lost, and more and more mature cells 
are killed. Also, the time after radiation that they die may lengthen from hours 
to a day or more. A good many young cortical neurons are killed by doses of 
several hundred r, but most of them escape for a while. Even after 800 r some 
members of the proliferative cell colony remain, and for the few days that an 
embryo so exposed may live, these residual cells actually go on proliferating 
brain or other neural structures as best they can. The little figures at the right 
in Fig. 1 emphasize the changing ratios of mature to immature cells as the fore- 
brain develops. At 13 days most of the primitive cells are involved in the pro- 
liferative cycle, and a cell no sooner divides into two postmitotic cells than the 
daughter cells enter the premitotic stage of the cycle again. This frenzied growth 
sub.sides by term, although a series of bursts of mitotic activity occurs in the fore- 
brain proliferative colonies between 13 days and birth, complicating the assess- 
ment of just which cells are radiosensitive. 

A detailed account of the brain malformations, especially in the cortex, and 
the mechanisms of their formation can be found in Hicks (1958), Hicks et al. 
(1954, 1959), and Hicks and D'Amato (1960a). The patterns of cortex of a rat 
irradiated with 200 r on day 13 is so completely difTerent from one irradiated 
on day 20, or day 16, that from the neurologic standpoint, lumping such animals 
together in behavioral experiments as "prenatally irradiated" would be absolutely 
meaningless. Considerable data on the patterns of malformation of the retinas 



160 



SUMMATION BY CHAIRMAN 



and the mechanisms involved in their formation, as well as similar information 
about the cerebellum, are now available (Hicks and D'Amato, 1960a, b) ; Hicks 
et al., 1959). There is no simple formula for the eye and retinal malformations, and 
radiation on any day from the 9th fetal to the 8th postnatal day presents its own 
problem, as Fig. 2 shows. Proliferation in the retina of the albino rat ceases about 
8 days after birth. Like the forebrain, the eye passes through a series of stages in 
which radiation does a variety of things. Restitution seems to be complete or 
almost complete, after the severe destruction that 200 r causes on day 13, and 
rosettes do not form. Rosettes are the cell balls that resemble distorted neural 
tubes and form from residual neuroepithelial cells after destruction of surrounding 
cells by radiation or other agents. Such distorted neuroepithelium continues to 
proliferate brain, cord, or retina, as the case may be. On day 20, for complex 
reasons, the retina is not very radiosensitive in the sense used here, yet on day 
19 or 21, mostly because there are greater numbers of sensitive migratory cells 
present, permanent changes occur. Of special interest for behavioral experiments 
is the great severity of the malformations that characterize the retinas of rats 
irradiated on the first few days after birth. From the morphogenetic standpoint, 
some immature bipolar cells are already present at birth, but when radiation 
destroys much of the adjacent layer of primitive migratory cells, these infant 
bipolar cells are suddenly stimulated to grow and sprout fibers, forming a preco- 
cious plc.xifonn zone. When the residual neuroepithelium begins to catch up, it 
spawns another layer of bipolar cells and itself forms rosettes as it differentiates 
into the rod cell layer. (Fig. 2). 

Malformations of the cerebellum are just as complex in their mechanisms as 
those of the cortex, thalamus, spinal cord, or retina. Figure 3 shows drawings of 

EFFECT5 OF 150to200p 
ON THE DEVELOPING RAT RETINA 




HEAD OEFGCTi 




RosETres 






REDuCfftrJ 





"I 

Fig. 2. The spectrum of eye malformations produced by 200 r 



2122 23 I 2 3fJS<i78 

PoiT |ta|NATftl. DATS 

Bipolar L^VER 



SUMMATION BY CHAIRMAN 



161 





ON THE 



ADULT CEReBELLUMS 
THAT RESULT FROM 200r> 
OM THE DAY 5HoWN 

IIttlE BRAIM5 SUOwJ 
5rA,C>E Ar Tirf\e OF 
RADIATION 



ON 7 He 3«o DAY 




On thc Ist 

(teSTHATAL PAV 



<:& 



Radiatiom om 

THE ZiiT FerAL DAY 



Fig. 3. The spectrum of cerebellar malformations produced by 200 r fror 
fetal to neonatal life. (.Adapted from Hicks and D'.\mato, 1960b.) 



late 



actual brains representing the gross patterns of cerebellar malformation in the 
albino rat that result from 200 r on the days indicated. In the gray rat, as in the 
mouse, the cerebellum is a little more advanced in its schedule of development 
than in the albino rat. The little brains, drawn to scale, show the fetal or neonatal 
brain as it was when radiation was given. Malformations induced at earlier stages 
are described by Hicks et al. (1959). In certain respects there is a front to back 
sequence of maturation of folia and a lesser center to lateral sequence, which is 
reflected in a corresponding sequence of deformations. To press this generality 
further would be misleading, because difTerent regions, and even parts of folia, 
wax and wane in their growth patterns. While the folial pattern is maturing, the 
cytologic characteristics of the cerebellum are also being unfolded, and the cyto- 
architectural malformations do not at all parallel the folial malformations. The 
malformations induced in late fetal life are characterized by a normal cyto- 
architecture, while those representing the end of the 1st week are characterized 
by an ectopic, extra granule cell layer, outside rather than deep to the Purkinje 
layer. Damage to the neuroepithelium, which in the developing cerebellum comes 
to lie on its surface instead of lining a ventricle, results at this stage in a preco- 
cious coarse growth of the Purkinje dendrites. This, we think, blocks the further 
migration of the primitive cells which would complete the granule layer. The 
late comers simply stop in the molecular layer. Irradiation on the first days after 



162 SUMMATION BY CHAIRMAN 

birth presents still other cytoarchitectural anomalies, including irregular ectopias 
of both granule and Purkinje cells. 

In summary, a certain amount of radiation delivered at given stages of early 
development results in unique patterns of response. The resultant morphologic 
patterns are distinctive for each stage, and it follows that the corresponding be- 
havior patterns must also be distinctive. 

References 

Hicks, S. P., 1958. Radiation as an experimental tool in mammalian developmental 
neurology. Physiol. Revs. 38, 337-356. 

Hicks, S. P., and D'Amato, C. J. 1960a. How to design £md build abnormal brains 
using radiation during development. In "Disorders of the Developing Nervous 
System," Charles C Thomas, Springfield, Illinois. 

Hicks, S. P., and D'Amato, C. J. 1960b. Malformation and regeneration of the mam- 
malian retina following experimental radiation. In "Symposium on Phakomatoses 
Cerebrale," (M. Feld, ed.), Salpetriere Hospital, Paris. 

Hicks, S. P., O'Brien, R. C, and Newcomb, E. C, 1954. Mechanisms of radiation 
anencephaly, anophthalmia and pituitary anomalies. Repair in the mammalian 
embryo. A.M.A. Arch. Pathol. 57, 363-378. 

Hicks, S. P., D'Amato, C. J., and Lowe, M. J. 1959. Development of the mammalian 
nervous system. I. Malformations of the brain, especially the cerebral cortex, in- 
duced in rats by radiation. II. Some mechanisms of the malformations of the cortex. 
/. Comp. Neurol. 113, 435-469. 

Hicks, S. P., D'Amato, C. J., and Joftes, D. L. 1961a. The nature of the radio- 
sensitive cells in the developing nervous system. In "Symposium on Effects of 
Radiation on the Nervous System" (B. Gross and V. Zeleny, eds. ). Intern. Atomic 
Energy Agency, Vienna, in press. 

Hicks, S. P., D'Amato, C. J., Coy, M. A., O'Brien, E. D., Thurston, J. M., and Joftes, 
D. L. 1961b. Some migratory cells in the developing nervous system studied by their 
radiosensitivity and tritiated thymidine uptake. Brookhaven Symposia in Biol. 14, 
in press. 

Sauer, F. C, 1935. Cellular structure of the neural tube. /. Comp. Neurol. 63, 12-23. 

Sauer, M. E., and Chittenden, A. C. 1959. Deoxyribonucleic acid content of cell 
nuclei in the neural tube of the chick embryo: Evidence for intermitotic migration 
of nuclei. Exptl. Cell Research. 16, 1-6. 

Sidman, R. L., 1961. Histogenesis of mouse retina studied with thymidine-Ha. In "The 
Structure of the Eye," Symposium, 7th Intern. Congr. Anatomists, New York, 1960 
(G. K. Smelser, ed.). p. 487. Academic Press, New York. 

Sidman, R. L., Miale, I. L.^ and Feder, N. 1959. Cell proliferation and migration 
in the primitive ependymal zone; an autoradiographic study of histogenesis in the 
nervous system. Exptl. Neurol. 1, 322-333. 

Watterson, R. L., 'Veneziano, P., and Bartha, A. 1956. Absence of a true germinal 
zone in neural tubes of young chick embryos as demonstarted by colchicine tech- 
nique (Abstract). Anat. Record 124, 379. 



PART 11 

Histopathological Changes Resulting 

from the Irradiation of the 

Nervous System 



Basic Problems in the Histopathology of 
Radiation of the Central Nervous System 



Orville T. Bailey 

University of Illinois College of Medicine, 
Chicago, Illinois 



This survey of the histopathologic chans^es resuhins; from radiation of the 
central nervous system will be confined to those detected by light microscopy 
in postnatal experimental animals and largely to the effects of gamma and 
roentgen radiation. Its purpose is to state some of the problems in tissue 
reaction following radiation as they are met by the neuropathologist and 
to illustrate rathei than offer solutions for these questions. 

Materials and Methods 

A series of experiments were carried out over many years in the Neuro- 
surgical Research Laboratory of the Children's Medical Center. Boston, 
under the direction of Dr. Franc D. Inoraham. in association with Drs. E. A. 
Bering, Jr.. R. L. McLaurin and others. Many results of these studies have 
been published Bailey ct al., 1957, 1958; Bering ft ai. 1955: McLaurin ct 
al., 1955 1. but the histologic findinss. especially in the spinal cord, ha\e not 
been described in detail. 

The experiments were of three types. In the first, tantalum'"-' wires co\- 
ered with polyethylene were inserted into the cerebral cortex of 40 monkeys 
(Macaca mulatta and Atelcs geoffroy). 1.5-2.0 mm posterior to the motor 
strip of the right cerebral hemisphere. The wires were removed after 2.5- 
4.770 r had been deli\ered. Monkeys were allowed to sur\ive from 2 hoius to 
33 months after completion of radiation. The polyethylene encasement was 
regarded as sufficient to pre\ent any tissue effects of beta radiation from the 
activated tantalum wire. 

In the second series of experiments, a piece of tantalum wire acti\ated in 
the atomic pile at Oak Ridge was encased in polyethylene and placed on the 
dorsal surface of the spinal cord or, in a few animals, beneath the skin over- 
lying the spine and fixed in place to the paravertebral fascia ! . Experiments 
were carried out in 18 Macaca mulatta monkeys with dosages varying from 

165 



166 ORVILLE T. BAILEY 

208 r to 55,000 r, measured at the center of the cord. Survival times were 
from 1 day to 36 months. 

The third series was concerned with the eflfects of roentgen radiation in 
17 Macaca mulatta monkeys receiving 138 r per minute to the lower dorsal 
region while under Pentothal or Nembutal anesthesia. Dosages varied from 
4,000 to 54,500 r, and survival times from 5 days to 18 weeks. In one addi- 
tional monkey, both Ta'^- and roentgen radiation were used. 

Time Factor in Tissue Responses 

The influence of time on the appearance, extent, and character of the 
histologic lesions produced by roentgen radiation has been recognized for 
over 50 years, the early studies being based on the skin of those engaged in 
therapeutic use of this agent (Wolbach, 1909). Using dogs Nemenow 
(1934a, b) studied physiologically in regard to conditioned reflexes, Lyman 
et al. (1933) have demonstrated the increase in the intensity of histologic 
lesions in the brain caused by roentgen radiation as the interval between the 
end of radiation and sacrifice is increased. This paper also contains a thor- 
ough review of the literature to 1933, as does tliat of Warren ( 1943). 

The eflfects of radiation in experimental animals has been turther consid- 
ered by Scholz ( 1934, 1935). In fully grown animals, the immediate reaction 
is not detectable microscopically, but if the animals survive from 4/2 weeks 
to 1 year alter ladiation, histologic lesions are stiiking. In a study of delayed 
lesions of brain and spinal cord in the dog, Davidoff ct al. (1938) pointed 
out that the rapidity with which clinical exidence of spinal cord injury ap- 
peared is proportional to the dosage and that disabilities in the monkeys tend 
to be progressive. The importance of time as a crucial factor in the appearance 
and interpretation of cerebral changes induced by roentgen radiation was 
carefully studied by Russell it al. ( 1949). They ha\e shown that with dosages 
of 2,850 r in rabbits no histologic changes were found before 82 days, but 
were present after that in all but one animal (of 7). Behavioral changes and 
abnormal neurologic signs did not appear until about 100 days after radia- 
tion, yet three rabbits killed before these changes appeared (82, 85, and 90 
days) showed well defined lesions. The brains of rabbits killed earlier than 
82 days after radiation showed no changes detectable by light microscopy. 
They also found that reducing the dose of radiation lengthened the latent 
interval. 

These and other substantial contributions have clearly established that the 
intensity of the histologic changes induced by roentgen radiation increase 
with time interval between radiation and sacrifice or death of the experi- 
mental animal. There are indications that the character of the lesions also 
alters with time. 



HISTOPATHOLOGY OF CNS RADIATION 



167 



Such proiiiession of lesions apparently does not take place in response to 
beta radiation, at least not to the same decree as with roentijen radiation 
(Campbell and Novick, 1949: Edwards and Bao;t>;. 192'3). 

More recently, interest in the neuropatholo2;y of radiation has tended to be 
focused on the acute phase of the reaction (Haymaker ft al., 1958; Voy;el 
ct al., 1958). By the use of relatively large doses of gamma radiation, it has 
been possible to characterize the acute lesion as seen by conventional 
methods of light microscopy as one of acute inflammatory changes and de- 
generati\e alterations in the cerebellum ( Vogel it al., 1958). These changes 
are the direct eflfect of radiation on the brain, since they do not appear when 
the whole body of the animal is radiated and the head shielded. While the 
acute changes are definite, they are mild in comparison with those which 
develop as time after radiation is increased. 

In personal studies, the experimental procedure precludes critical evalua- 
tion ot the acute phases of radiation reaction. 1 antalum'''- deli\ers radiation 
at a rate such that se\eral days are recjuired to accumulate the dosages 
necessary. When Ta'^- wire, shielded with polyethylene to prevent beta 
radiation fiom complicating the picture, is inserted into the cerebrum of 




Fig. 1. tloronal section of monkey brain, showing representati\e lesion induced by 
insertion of Ta"'" wire. Dosage. 600 r in 5^ days. Left hemiplegia developed aftei 
3 months. Sacrifice 1 year after radiation. Lesion measured 7x4 mm. 



168 ORVILLE T. BAILEY 

monkeys posterior to the motor strip, an area of necrosis is produced (Fig. 
1 ) . Observation of such animals gives some indication of progression in the 
lesions. Hemiparesis which developed in 6 of 21 monkeys did not appear 
until 3 weeks to 20 months after radiation, the animals previously being 
neurologically normal (Bailey ct al., 1958). 

Electroencephalograms a few days after radiation showed decreased voltage 
and some slow waves on the radiated side, while on the opposite side there 
was a predominant frecjuency faster than the one before radiation. This 
condition remained and was most prominent 6 to 8 weeks after radiation. 
The EEG pattern then reverted almost to normal. At longer intervals, slow 
waves again appeared on the radiated side, and many records demonstrated 
voltage asymmetry with decreased voltage on that side. Two years after 
radiation, 2 animals developed runs of spikes and fast activity localized to 
the region of radiation. One of these developed generalized clonic and tonic 
seizures 30 months after completion of radiation (Bailey et al., 1958). There 
was thus some EEG evidence of progression. These results are in fair agree- 
ment with those of Ross ct al. ( 1954), though the conditions of radiation are 
so different that direct comparison is difficult. 

The histologic changes in these animals became more striking as the 
interval between radiation and sacrifice was lengthened. There was more 
evidence of irregular streaks of injury extending out from the necrotic region 
in which the Ta^^- wire had originally been placed (Fig. 1 ) . 

Studies of radiation efTects in the spinal cord of experimental animals 
have not been numerous (Cairns and Fulton, 1930; DavidofF ct al., 1938; 
McLaurin ct al, 1955; Pendergrass ct al, 1922; Peyton, 1934; Sicard and 
Bauer, 1907). However, the spinal cord has pro\ed a \ery favorable area 
for the study of radiation efTects. 

In personal studies, clinical observations ot monkeys gave .some evidence 
of the time factor as an important consideration. With Ta^""- radiation of 18 
monkeys, 2 developed transitory paraplegia with complete recovery, 6 perma- 
nent paraplegia, and 1 early paraplegia, then recovery followed by permanent 
paraplegia after 6 weeks. When roentgen radiation was used, 6 of 1 7 animals 
developed paraplegia. In some monkeys dying with complete paraplegia 
within a few days after either form of radiation, there were no histologic 
changes or only scattered vacuolation of myelin in white fiber tracts. In view 
of the striking alterations described in monkeys surviving for long periods 
after radiation, there is good evidence that the histologic changes become 
progressively more obvious, at least to light microscopy, as the inter\al be- 
tween completion of radiation and sacrifice is lengthened. 

The time factor thus becomes a dominant consideration in defining dosage 
effective in causing histologic change and especially in determining the ulti- 
mate effect on a living organism subjected to ionizing radiation. 



HISTOPATHOLOGY OF CNS RADIATION 169 

High Energy versus Low Energy Radiation 

In pre\ious experiments, the tolerance of the spinal cord of Macaca 
mulatta for gamma radiation was approximately 135 r per hour and 
about 125 kv r for roentgen radiation ( McLaurin et al., 1955). Radiation 
from activated Ta has a much higher energy than the roentgen radiation 
used in our studies. These results suggest that, under the experimental condi- 
tions used, low energy radiation is slightly more effective in producing para- 
plegia than high energy radiation. This agrees with the findings of Arnold 
et al. (1954a). However, other investigators (Hicks it al., 1956) ha\e found 
that central nervous system tissues are more sensiti\e to high energy radiation. 

It seems difficult to reconcile these dixergent results. Among the studies, 
there are data from difTerent animals, and sources of radiation also \ary 
somewhat. E\en so. when both types of radiation ha\e been carried out in the 
same laboratory under conditions as nearly controlled as possible, contradic- 
tions in results remain. The situation is no clearer in regard to the sensiti\ity 
of tissues outside the central nervous system. The cjuestion remains a signifi- 
cant one ior finthei' inxestigation. 

Effect of Intensity of Radiation 

The intensity of radiation has emertied as an important factor in the tissue 
response in the nervous system (Hicks et al., 1956; McLaurin et al., 1955). 
Intensity as a factor in other organs has produced more equixocal results 
(Brunschwig and Perry, 1936; Pack and Quimby, 1932). 

The effect of the rate at which a given dose of radiation is administered is 
strikingly demonstrated by 2 monkeys in personal material. Each received the 
equivalent of 55,000 r of gamma radiation to the spinal cord, at the rate of 
4.000 r per day in one and at 1,870 r per day in the other. The first animal 
developed a flaccid paralysis in the 2nd week after radiation, which pro- 
gressed steadily in severity until death at 2 months. The second monkey 
showed no neurologic deficit until its death irom an independent cause 4 
months after completion of radiation. 

In the series in general, it was found that 7.500 r as a single dose were 
required to produce paraplegia, but two doses of 5.000 r were necessary 
(McLaurin (/ al.. 1955). These results are somewhat diflferent from those of 
Davidoflf <7 al. 1938). who found 5.000 r sufficient to cause paraplegia. 
However, they used onlv 1 animal at that dosage. 

Differences in Tissue Reaction with Age 

There is e\idence that changes in young animals are different Irom those in 
adults of the same species. In young animals, smaller doses of radiation are 
required to produce behavioral and histologic changes in the brain than in 



170 ORVILLE T. BAILEY 

fully grown ones, and the period between radiation and overt tissue degen- 
eration is shortened (Clemente et al., 1960; Mogilnitzky and Podljaschuk, 
1928, 1929; Scholz, 1934, 1935; Yamazaki ct al, 1960; Ziminern and 
Chavany, 1931 ) . 

Clemente et al. (1960) found that as little as 125 r of roentgen radiation 
to the head may result in microcephaly and cataracts if gi\en to rats 8 hours 
old, while 300 r produces abnormal neurologic signs and histologic changes 
in most rats at 1 day and 4 days of age, but not at 7 days. They feel that 
resistance of the brain to radiation becomes significantly increased toward 
the end of the 2nd week of postnatal life. Their histologic studies indicate a 
high degree of correlation between abnormal neurologic signs and histologic 
lesions in these immature mice. While some changes, predominantly vascular, 
are found in rats sacrificed at 48 72 hours, they state, "It seemed as though 
processes were under way which would result in larger necrotic sites, espe- 
cially in rats administered 1,000 r, had these animals been allowed to live 
for longer periods. This assumption seems especially \alid since other animals 
radiated at the same postnatal time and sacrificed 1 to 14 months later 
showed larger lesions in the brain." (Clemente ct al., 1960). 

This work is in some way reminiscent of Hicks's ( 1953, 1954) results in 
antenatal development. The occiurence of cerebellar changes and micro- 
cephaly is in accord with his timetable. The eye defects are of a different 
type from those Hicks produced by radiation early in pregnancy. 

For this reason, the results of Clemente it al. (1960: Yamazaki et al., 
1960) in some ways may correspond to late prenatal radiation in certain 
other species of animals in which brain development is more advanced at 
birth. However, they are of particular interest because they are quantitative 
studies in brains with no, or restricted, regenerative capacity. They are also 
in accord with results in puppies reported by others (Lyman et al., 1933; 
Scholz, 1934,1935). 

It seems reasonable to conclude that the nervous system of young animals 
is considerably more sensitive to radiation than that of adult animals of the 
same species, that the time inter\al between radiation and o\ert histologic 
evidence of degenerative changes is less in young animals, and that abnormal 
neurologic signs, behavioral changes, and histologic lesions are regularly 
produced with lower dosages of radiation in young animals than in old 
ones. The particular alterations that accompany maturation in the neuron 
so that it changes its response to ionizing radiation in these ways remains an 
important problem. 

Vascular Responses to Radiation 

One characteristic of the reaction to radiation in all tissues is the develop- 
ment of degenerative and occlusive changes in blood \essels. The central 



HISTOPATHOLOGY OF CNS RADIATION 171 

nervous system is no exception. There is general agreement that aherations 
in vessel walls are found in all phases of radiation reaction in the brain and 
spinal cord. The sequences in\olved and the importance of these changes in 
the total picture of radiation injury are still not entirely established. 

Clemente ct al. (1960) consider the earliest and most constant vascular 
reaction to be a swelling- of the cytoplasm of endothelial cells and an increase 
in the intensity of basic staining in the nucleus. They feel that this process 
may be reversible or arrested or may progress to capillary rupture and in- 
flammatoiy cell infiltration. Polymorphonuclear leucocytes appear soon after 
radiation, as early as 6 hours (Clemente and Hoist, 1954), and tend to dis- 
appear after about fi\e days (Clemente and Hoist, 1954; Haymaker ct al.. 
1958). Rachmanow 1926) has demonstrated accumulation of trypan blue 
in the endothelial cells at this stage. These early reactions are most con- 
spicuous in capillaries, where they may be associated with detectable necrosis 
of the wall. Such lesions of capillaries would accoimt for the frequent occur- 
rence of minute hemorrhages in the acute phase of radiation reaction 
(Alquier and Faure-Beaulieu. 1909; Clemente and Hoist. 1954; Haymaker 
rt al., 1958 1 . In animals surviving longer after radiation, hemosiderin deposits 
mark the location of pre\ious small hemorrhages i Fig. 1 ) . Larger hemor- 
rhages rarely ha\e been described (Rachmanow, 1926: Scholz, 1935) and 
usually in animals surviving 3 or 4 weeks after radiation. C'apillary changes 
may be related to the increased permeability of the blood-brain barrier 
(Clemente and Hoist. 1954; Mogilnitzki and Podljaschuk, 1930) and per- 
haps less directly to brain swelling noted by sexeral authors ( Gerstner rt al., 
1954; Ross <■? a/., 1953). 

Damage to larger \essels becomes more obvious at longer inter\als after 
radiation. Severe necrosis with disruption of vessel walls occurs in the brain, 
spinal cord and meninges. This is accompanied by cellular infiltration which 
presumably is at first polymorphonuclear, but at the stage usually seen is 
predominantly lymphocytic with a component of macrophages. In well 
marked examples, few vestiges of the structure of the \asculai wall remain 
(Fig. 2). The line of the endothelium and its basement membrane are sepa- 
rated from an adventitia which is hea\ily infiltrated with inflammatory cells, 
but is not necrotic i McLaurin ct al.. 1955). With time, there is repair and 
reshaping of the vascular wall, the media retaining its form, but being com- 
posed mostly or entirely of fibrous tissue i Fig. 3 i. Vascular occlusion is often 
completed by an organi"ed thrombus filling the lumen. This process as a 
general phenomenon of \ascular repair has certain analogies with the repair 
of vessels in hypersensiti\ity reactions i Hawn and Janeway. 1947, especially 
their Fig. 12). Such similarities should not be interpreted as indicating a 
related pathogenesis, but as reparatixe phenomena in ncciosis of similar 
distribution. 



172 



ORVILLE T. BAILEY 




Fig. 2. Vascular lesion in small leptomeningeal vessel near an area of myelomalacia. 
There is heavy inflammatory cellular infiltration of the necrotic wall, but the en- 
dothelial layer appears intact. Gallocyanin-van Gieson X250. Dosage 5,606 r Ta'*" in 
3 months. Paraplegia at end of radiation: death 1 week later. Autopsy: myelomalacia 
10th thoracic to 2nd lumbar segments. 



There is at least one other type of vascular occlusion in the central nervous 
system of chronic animals. This occurs when there has been further necrosis 
of the collagenous tissue produced in repair followed by secondary collagen- 
ous response, eventually producing enlarged, bizarre vascular channels with 
tiny lumina (Fig. 4) or none at all. 

These striking changes in vessel walls of chronic animals are not nearly so 
widespread in the area of radiation as in the vasculitis of smaller vessels in 
the acute phase. There is considerable evidence that the late vascular changes 
are segmental. Hence more vessels may have an occluded segment at some 
point than would be inferred from a single microscopic section or even from 
several sections of one tissue block. 



HISTOPATHOLOGY OF CNS RADIATIOxX 



173 




Fig. 3. Vascular lesion in small leptomeningeal vessel near. an area of myelomalacia. 
End result of radiation change with fibrosis of wall and complete obliteration of the 
lumen. Hematoxylin-eosin X250. Dosage 4,440 r Ta'''"' in 29 hours. No neurologic 
deficit. Sacrifice 4 months after radiation. Autopsy: partial myelomalacia at 12th 
thoracic and 1st lumbar segments. 

One of the most perple.xins; problems in the histopatholo<;y of radiation 
reactions is the relation of such lesions to other chano;es induced by this 
agent. As long ago as 1921, Bagg stated that x-ray injury to the brain is 
secondary to vascular change. Since that time, workers have been di\ided 
as to whether the changes in the parenchyma are ischemic and infarctive or 
whether they are direct eflfects of ionizing radiation without mediation 
through vascidar mechanisms. The opinion of the author, based on personal 
material, is in agreement with Arnold's et al. ( 1954b) that the effects on the 
nervous system are direct effects. The occlusion of vessels could account for 
areas of complete infarction in the region of their distribution. Such effects, 
however, are only a minor part of the response of the central ner\ous system 
to ionizins radiation. 



Effects of Radiation on the Neuron 

Largely through the work of Hicks (1953, 1954), it has become generally 
recognized that the developing neuron is highly susceptible to ionizing radia- 



174 



ORVILLE T. BAILEY 




Fig. 4. Marked vascular Irsions after intracerebral implantation of Ta""". Weil's 
method X30. Dosage 553 r in 13 days; sacrifice 16 days after completion of radiation. 
No neurologic deficit. Lesion measured 8 mm in diameter. 



tion. Resistance to such injury incieases in older animals. There is less gen- 
eral agreement as to whether the adult neurons are directly injured or 
whether the nerve degeneration is secondary to vascular lesions. 

In the acute phase of the radiation reaction to gamma and roentgen rays. 
the changes visible by light microscopy in the nerve cells are not striking. 
Alvord and Brace ( 1957) found that there is pyknosis of granule cells in the 
cerebellum, which is probably reversible and coincides with a period of 
clinical neiuologic dysfimction. This is maximal at 8 hoius after 7,500 r of 
whole body radiation. Similar changes are produced if only the hindbrain 
and cerebellar regions are radiated, but no alterations occur when this area 
is shielded. The same type of pyknosis in the cerebellar granule cells has been 
produced by Vogel et al. ( 1958) using cobalt''" ( 10,000 r), and they also feel 
that this effect is transient and reversible. The results of Hicks it al. ( 1956) 
are in agreement. In none ot these studies is there evidence of vascular 



HISTOPATHOLOGY OF CNS RADIATION 175 

changes in close association with the cerebellar lesions: the alterations in the 
grannie cells are regarded as direct effects of radiation. 

Campbell ct al. (1946), on the other hand. ha\e found early changes in 
Purkinje cells. Haymaker et al. { 1958) have noted somewhat similar changes 
in Purkinje cells, but also have found these types of alteration in a control 
monkey. Nerve cell bodies in other regions of the ner\ous system are little 
affected in the acute phase of the radiation reaction. Brownson (1960), in 
determining whether any effects on these structures can be detected by 
changes in the perineuronal satellite cells, found no statistically significant 
alteration in neuron-neuroglia relationship ot the cerebral cortex alter 
1,600 r. 

Though less extensi\ely studied, beta radiation apparently exerts an effect 
directly on the nerve cells (Campbell and No\ick. 1949). 

Delayed necrosis of the para\entricular and supraoptic nuclei of the hypo- 
thalamus has been demonstrated by Arnold 1 1 al. ( 1954b) . This is a specific 
effect and is produced by doses of 3.000 r or less, no radioselectivity being 
noted with larger doses. CMemente and Hoist (1954) also have encountered 
consistent inxohement of the hypothalamus. 

There is a high degree of radioselectivity for the white matter ' Arnold 
et al., 1954b I. The neuronal necrosis becomes progressively more e\ident as 
the interval between radiation and sacrifice of the animal is lengthened. 4'his 
delayed reaction is one of the most striking differences in histology between 
the acute and late phases of radiation change. 

An extensixe literatiuc is in almost complete agreement that the brainstem 
is the most sensiti\e region (Arnold et al.. 1954a, b. c: Colwell and Glad- 
stone. 1937: Demel. 1926: Ellinger. 1942: Ellinger and Davison. 1942: 
Mogilnitzky and Podljaschuk. 1928). Hicks and Montgomery (1952) also 
describe special sensiti\ity in parts of the ""oltactory brain."" At least in the 
dosages generallv used, the injurv tf) the brainstem in\ol\es both white and 
gray matter. In fact. C'.olwell and Gladstone i 1937 ) emphasize ner\e cell 
changes in these regions and in the central gray matter of the cerebrum. 

Personal material, using the monkey spinal cord, is in accord with the 
literature cited in regard to late effects on the neuron. The nerve cell bodies 
in both anterior and posterior horns show no detectable changes after extra- 
dural application of Ta'""-' or roentgen ladiation. except when in an area of 
total necrosis. 

Nerve fiber necrosis in the posterior portion of the spinal Cf:)rd is iiregular. 
but widely distributed, and extends little beyond the immediate zone ot 
radiation, either with Ta'^- or roentgen radiation. In the monkeys sacrificed 
in less than 2 weeks after completion of radiation, the only change found was 
occasional myelin degeneration in isolated segments. In later stages, the 
<'xtent of mvelin defeneration is somewhat greater than that of demonstrable 



176 



ORVILLE T. BAILEY 




Fig. 5. Longitudinal section through posterior (.oluinns ol spinal cord to show 
myelin sheath degeneration. Gallocyanin-van Gieson X250. Dosage 6,220 r Ta"" in 
43/2 hours. Slight weakness of legs and loss of sphincter control 3 days after radiation: 
gradual recovery over 10 weeks; monkey then normal 1 month; gradual onset of 
paraparesis, persisting until sacrifice 9 months after radiation. Compare with Fig. 6. 



neuron disintegration, a finding in agreement with Reynolds (1946), who 
compares this effect with that obtained with multilayer films of lecithin 
radiated on a water surface. Doses as low as 600 r destroy the normal molecu- 
lar arrangement. 

In embedded sections, this myelin degeneration appears as scattered vacu- 
oles (Fig. 5), corresponding in distribution with droplets stained with oil 
red O (Fig. 6). The areas which fail to take such stains for the demonstra- 
tion of myelin as Weil's method are considerably larger than shown by the 
other two techniques. Reasons for this difference are not clear. 

The nerve fibers themselves are interrupted by irregular zones of necrosis 
affecting individual fibers (Fig. 7). Adjacent nerve fiber segments are 
swollen or partially fragmented. While the distribution of such injured nerve 
fibers corresponds closely to the area of radiation, it is possible with smaller 



HISTOPATHOLOGY OF CNS RADIATION 



177 




I'u:. h. Longitudinal section through posterior cohnnns of spinal eord stained with 
oil red 0, X250, to show the similarity in distribution of droplets stained by this 
method and vacuoles seen in Gallocyanin-van Gieson (Fig. 5). Same monkey as Fig. 5. 



doses or at the edye of the zone of reaction to find patches of degenerated 
fibers ( Fis;. 8 ) . 

The distribution, time of demonstration by histologic methods, and the 
relation to vascular changes all support the vievs- that the effects of gamma 
and roentgen radiation are direct effects on the parenchyma of the central 
nervous system and are not mediated through vascular insufficiency. The 
long latent period before these changes become apparent to the light micro- 
scopist is a time when the more quickly demonstrable vascular changes 
dominate the histologic picture. But this does not imply that the vascular 
lesions are causati\e. It suggests that changes at a submicroscopic, possibly 
molecular, le\el have been initiated at the time of radiation, changes which 
are compatible with preservation of morphologic structure for weeks or 
months. An endpoint must be reached not only before the light microscope 
can detect the changes, but also before the cellular sequences of repair are 
initiated. 



178 



ORVILLE T. BAILEY 







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J^^ -i-^' - ;-;:,• Ar>. ^^^ ••--'^iVwlr" "Jr 






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HISTOPATHOLOGY OF CNS RADIATION 179 

There is evidence ( Bailey <"/ «/., 1958: Gerstner c/ ai, 1955; McLaurin ct ai, 
1955; Yamazaki ft ai, 1960) that in this latent period there may be func- 
tional changes related to the radiation injury before histologic change is 
established. The demonstration and definition of this latent interval is the 
role of the light microscopist ; the electron microscopist and chemist must 
elucidate the processes durino- that time. Such enzyme studies as that of 
Clammermeyer and Haymaker ( 1958) are promisino;. 

Effects of Radiation on Glia 

Arnold and P. liailey (1954) have pointed out that the response to 
\-radiation involves damage to all types of adult glial cells, depending on the 
total dose of radiation, the intensity of dose administration, the uniformitv 
of dose distribution, and the time inter\als between radiation and sacrifice of 
the animals. 

The oligodendroglia are early and se\erely affected, with swelling of the 
cytoplasm (Arnold and Bailey, 1954) followed by pyknosis and disintegra- 
tion of the cells (Hicks and Montgomery, 1952; Hicks ft ai, 1956). In \iew 
of the generally accepted role of oligodendroglia in the maintenance of the 
myelin sheath, this finding is of considerable interest because of the relatively 
early disintegration of myelin in radiated areas (Hicks ft <?/., 1956) and the 
severity of radiation damage at the period of acti\e myelination (C'lemente 
ft a!., 1960). 

There is little, if any, lormation of compound granular corpuscles in areas 
of necrosis (Arnold and Bailey, 1954), indicating inhibition or destruction of 
microglia. 

A similar but more striking eflect is exerted on the astrocytes, lliey at first 
swell, then fragment, and by 4 to 6 weeks after radiation the area is de\oid 
of astrocytes ( Arnold and Bailey, 1 954 l . Only after many months is there 
resumption of the expected astrocytic proliferation in repair of necrotic 
areas. 



Fig. 7. Patchy loss of myelin in dorsal spinocerebellar fasciculus, suggesting direct 
injury not mediated through blood vessel changes. Weil's method X30. Monkey re- 
ceived 12,048 r Ta^"" in 67 hours. X-ra\- 10.000 r in 3 days given 5 months after 
completion of Ta'"" radiation. Complete paraplegia 48 hours after Ta""" radiation: 
partial recovery in 2 months; paraplegia again complete after x-radiation. Sacrifice 6 
weeks after completion of x-radiation. See also Fig. 1 1. 

Fig. 8. Degeneration and fragmentation of nerve fibers in posterior columns of 
spinal cord. Hortega's silver carbonate X350. Dosage 10,000 r x-radiation in 2 days. 
Clomplete paraplegia 36 hours after completion of radiation. Sacrifice 5 days after 
radiation. 



180 ORVILLE T. BAILEY 

-Campbell and Novick (1949) have found that astrocytes are most suscep- 
tible to beta radiation, oligodendroglia and microglia being highly resistant. 
Haymaker et al. (1958), using barium^'*"-lanthanum^^'^' as the source of 
gamma radiation obtained results which diflfered from those of Arnold and 
P. Baifey ( 1954) with roentgen radiation. They conclude that alterations in 
glial cells are relatively inconspicuous. It is difficult to reconcile these two 
observations. 

Personal studies of material using Ta^*"- as a source of gamma radiation 
give results entirely in agreement with those obtained by Arnold and P. 
Bailey (1954). While the experimental procedure was not suitable for the 
study of early changes, the specimens studied at relatively short intervals 
after radiation showed disintegration of oligodendroglia coextensive with 
myelin loss and almost complete, if not total, inhibition of compound gran- 
ular corpuscle formation. The disintegration of astrocytes extended through- 
out the area of maximum radiation effect and for a short distance beyond 
where neurons were demonstrably afTected. In later stages, the inhibition of 
the expected astrocytic proliferation was a striking feature of the radiation 
response, but at the longest internals (up to 3 years) after radiation, brisk 
astrocytosis was again resumed. It is difficult to be precise in regard to the 
time when astrocytes begin to respond in the expected way as a part of the 
sequences of repair. Arnold and P. Bailey (1954) placed it at "many 
months." Our material can make this interval no more exact. 

Behavior of Collagen in Radiated Areas and its Significance in 
Reparative Sequences 

Little attention has been paid to the effects of ionizing radiation on the 
collagen of the central nervous system parenchyma. Confined as it is under 
normal conditions to the region of blood vessels, it is relatively inconspicuous. 
However, it is important in the normal nei'vous system and participates in 
reparative sequences. 

In personal material, the growth of collagen is the dominant feature of 
the initial tissue response in brain and spinal cord areas where radiation has 
produced total necrosis. This type of lesion has been studied to best advan- 
tage in the spinal cord where no operative procedure had been carried out 
within the parenchyma. Necroses studied a few weeks or months after radia- 
tion fail to show any evidence of cellular repair (Fig. 9), a finding in agree- 
ment with Arnold and P. Bailey (1954). Some of these necroses extend 
completely across the spinal cord (Fig. 9) and occasionally a part of the 
necrotic material protrudes as small elevations into the subarachnoid space 
in small zones of partial destruction of the pia (Fig. 10). No instances of 
complete necrosis of the pia or escape of necrotic material into the subarach- 



HISTOPATHOLOGY OF CNS RADIATION 



181 










Fig. 9. Luntjitudinal station ol spinal ( urd lo sliow the junction of a zone of total 
radiation necrosis with surviving spinal cord tissue. Weil's method X40. Dosage 
54,500 r x-radiation given in 23 days. On the day after completon of radiation, the 
monkey developed paraparesis which progressed to complete paraplegia in a few 
hours. Sacrifice 1 week after radiation. .Autopsy showed a constriction in the spinal 
cord at the 7th and 8th thoracic segments. See also Fig. 10. 



noid space have been encountered. Other necroses are smaller and in\olve 
localized regions of white matter. 

When repair begins to take place after some months, the collagen pro- 
liferates long before the inhibition of astrocytes has ceased. Compound 
granular corpuscles make their first appearance at about the time that 
production of collagen is resumed. 

The result of these alterations in the growth capacities of the cells usually 
involved in repair of central nervous system lesions is a localized, walled-ofT 



182 



ORVILLE T. BAILEY 




Fig. 10. Longitudinal section of spinal cord, showing herniation of necrotic tissue 
in an area of damage to the pia. Gallocyanin-van Gieson Xl50. Same monkey as 
Fig. 9. 



area. The layer next to the surviving spinal cord tissue is composed of colla- 
genous tissue with its fibers oriented about the central necrotic material, now 
fragmented and amorphous (Fig. 11). Compound granular corpuscles are 
found in the meshes of the collagenous tissue and in the necrotic central 
region, but astrocytes are absent. The lesion at this stage is somewhat more 
reminiscent of repair of necrosis in tissue outside the central nervous system 
than within it. 

In monkeys sacrificed at still longer intervals (usually 8 months and 
longer), proliferation of astrocytes is conspicuous outside the zone described, 
but the collagenous scar remains unpenetrated by astrocytes in its central 
dense zone. 

Further opportunities to study the behavior of collagen are aflForded by the 
monkeys in which Ta^*"" activated wire has been inserted into the corona 
radiata. It is difficult to evaluate collagen behavior in or near the point of 



HISTOPATHOLOGY OF CNS RADIATION 



183 



7 -:^.-''>«i 











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^^:'$.^M^-:: 









Fig. 11. Area of necrosis in the spinal cord with connccti\e tissue proliferation and 
coni]30i.uid granular corpuscle formation. l)ut without astrocytosis. Gallocyanin-\an 
Gieson X 2'_'5. Same monkey as Fi-^. 7. 

insertion because of tlie possible participation of meningeal collagenous 
tissue. However, in the depths of the lesions at a few weeks or months after 
radiation, the same proliferation of collagenous tissue without admixture ol 
astrocytes is present (Fig. 12). Occasionally, it can be seen that collagenous 
tissue is growing into regions of fibrin deposition (Fig. 12). but usually such 
a relationship cannot be established. When astrocytic proliferation is re- 
sumed, these areas of collagenous scar remain lor the most part intact. 

An attempt has been made to hnd out whether there is secondary degen- 
eration of the collagen and regrowth of new collagen, as has been described 
in radiation reactions in the skin (Wolbach, 1909). Occasionally, hyalinized 
collagen fibers into which secondary proliferation of collagenous tissue has 
taken place, can be seen, but this is uncommon. Some vessel walls (Fig. 4) 
have appearances suggestive of repeated collagenous repair. 



184 



ORVILLE T. BAILEY 




Fig. 12. Clollagen production in the depths of an area of radiation necrosis at a 
stage before proliferation of astrocytes has begun. Gallocyanin-van Gieson X250. 
Dosage 600 r Ta'"" in 8 days to right corona radiata. Mild hemiparesis developed. 
Sacrifice 20 days after completion of radiation. Area of necrosis 7 mm in diameter. 



Radiation Reactions in Other Structures 



In the acute phase of the radiation reaction in menins^es, inflammatory 
changes with polymorphonuclear leucocytic infiltration are conspicuous with 
doses of 300 r or larger. The process is localized with smaller doses, but tends 
to spread more widely as the dose is increased. At longer intervals after 
radiation, the cellular infiltrate becomes mononuclear with some connective 
tissue proliferation. Vascular lesions occur within the meninges and undergo 
the same secjuences as those within the brain parenchyma (Clemente ct al., 
1960; Haymaker et al., 1958; personal material). 

In the choroid plexus inflammatory changes similar to those in the menin- 
ges are present in the acute phase, occasionally accompanied by small hemor- 
rhages. The end result of these changes is a small fibrous scar. All portions of 



HISTOPATHOLOGY OF CNS RADIATION 185 

the choroid plexus are about equally affected (Clemente et al., 1960; Hay- 
maker et al., 1958) . 

Dilatation of the ventricular system has been seen after radiation 
(Clemente et al, 1960; Demel, 1926), but this is variable from animal to 
animal. It is greatest in young animals. 

In the pituitaiy, both anterior and posterior lobes are severely damaged in 
head or whole-body radiation f Haymaker et al, 1958; Mogilnitzky and 
Podljaschuk, 1928: Vogel et al., 1958). 



Comment 

In spite of numerous disagreements in the results of various workers, a 
general pattern of degeneration and repair is beginning to be defined in the 
tissue reactions of the central nervous system to gamma and roentgen radia- 
tion. It is quite possible that further advances can be made by particular 
study of points where contradictions in results now exist. 

The series of studies re\iewed ha\e been carried out on widely dixergent 
species of animals, ranging from goldfish to monkey. It is not certain how 
many of the contradictory results are dependent on species variation or to 
what characteristics of indi'.idual species these differences arc related. The 
demonstration of certain constant features in a wide range of experimental 
animals suggests that species differences may be more related to details than 
to the general pattern of response. The high degree of sensitivity of the 
brainstem in animals far apart in the phylogenetic scale is an instance in 
point. 

In nearly all experiments re\iewed and in personal material, there are 
e\en more puzzling variations from animal to animal when radiation source, 
experimental conditions, and other technical aspects have been kept as 
constant as possible. Special studies of the animals which are particularly 
sensitive or resistant in a given set of experimental conditions may suggest 
factors not well recognized. 

Some such factors may involve the biologic state of the animal at the time 
of radiation. Rugh ( 1958) has stated, "Probably the most effective physical 
factors which influence irradiation sensitivity at any biological level are: 
(a) state of hydration, (b) degree of oxygenation, and (c) amount of 
activity or movement." The abolition of early cerebellar effects by bar- 
biturate anesthesia, as demonstrated by Alvord and Brace (1957'i, may be 
related to such factors. The apparently simple question of what is the 
minimum dose of radiation which produces damage to the central nervous 
system is actually one of great complexity. 



186 ORVILLE T. BAILEY 

Summary 

A review of the literature related to the effects of gamma and roentgen 
radiation of the central nervous system is presented and compared with 
personal material, with a few comments on the effects of beta radiation. 

The reaction of the central nervous system hours or a few days after radia- 
tion is an acute inflammatory one. dominated by \asculitis, meningitis, and 
choroid plexitis. Regressive changes, apparently mostly reversible, are present 
in the granide cells of the cerebellum. 

Young animals are more susceptible to radiation damage than adults, but 
histologic effects can be produced at any age with sufficient dosage. 

Different and more extensive degenerative processes become evident as the 
time interval between radiation and sacrifice is lengthened. This interval can 
be reduced by increasing the dose of radiation, increasing the intensity of 
radiation, and possibly by other factors. 

In late stage of radiation reaction, there is extensive damage to neurons, 
with selectixity for the white matter and particidar sensitivity of the brain 
stem and hypothalamus. Degeneratixe changes in blood vessels, sometimes 
with complete occlusion, can be demonstrated at this stage. 

The effects on neurons are considered by most, but not all, workers to be 
direct effects and not mediated through \ascular insufficiency. 

All types of glia show degenerative changes after radiation, and there is a 
prolonged inhibition of glial response in repair. Proliferation of collagenous 
tissue is important in the first stages of repair. C'ompound granular corpuscle 
formation is resumed before astrocytic proliferation begins. Some months 
after radiation, astrocytosis becomes exuberant. 

Despite many contradictions in results and interpretation, a basic pattern 
of degeneration and repair in response to gamma and roentgen radiation is 
becoming apparent by light microscopy. The most striking single featme is 
the progressive increase in evidence of damage as time after radiation is 
lengthened. It is impossible to predict end results from any characteristics of 
the acute response. Investigations by all techniques will be recjuired to 
explain the processes going on in the interval, and studies of this type may- 
well have biologic implications beyond the field of radiation pathology. 

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Exptl. Neurol. 16, 3-17. 



HISTOPATHOLOGY OF CNS RADIATION 187 

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Clemente, C. D.. and Hoist, E. A. 1954. Pathological changes in neurons, neuroglia. 

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Colwell. H. .\.. and Gladstone. R. J. 1937. On some histological changes produced in 

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188 ORVILLE T. BAILEY 

Demel, R. 1926. Tierversuchc mit der Rontgenbcstrahlung des Cerebrum. Strahlen- 

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Vogel, F. S. 1958. The effects of barium 140 — lanthanum 140 (gamma) radiation 

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Hicks, S. P. 1953. Developmental malformations produced by radiation. A timetable 

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central nervous system. Rt-sidts of large doses on the brains of aduh dogs. A.M. A. 

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HISTOPATHOLOGY OF CNS RADIATION 189 

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Sequence of X-Radiation Damage in 
Mouse Cerebellum 



NORBERT SCHUMMELFEDER 

Institute of Pathology 
University of Bonn, Germany 



Since the time of Brunner and Schwartz 1918: Brunner, 1920, 1921), 
who in 1918 were the first to observe that the cerebellar o;ranule cells of 
young- dogs and cats could be easily injured by x-radiation, little attention 
was directed to the radioxulnerability of this part of the brain until the past 
few years. Our work in this field was concerned with the effect of x-rays 
on the cerebellum of mice ( Schiimmelfeder 1957, 1959a, b; Schiimmel- 
feder ct al, 1957; Krogh and Bergeder, 1957). We used single x-ray doses 
and studied the irradiated tissue by morphologic, histochemical, and fluo- 
rescence techniques. The x-ray dosage ranged from 250 to 60,000 r. Fields 
of the cerebellum .3 X '^ "ini and 0.5 X 2 mm were irradiated by a half-wave 
x-ray unit (50 kv, 20 ma. focal distance 6 cm. 0.12 mm Al filter) at 3,000 
r per min to the surface of the cerebellum. The irradiated animals were 
sacrificed at interxals up to 6 months. 

Observations 

Morphologically demonstrable radiation effects were seen in the range of 
2,000 to 60,000 r. T\w latent period between irradiation and the first mor- 
phologic changes decreased correspondingly. At 2,000 r, damage was first 
noted at the end of 4 months, while at 60,000 r, changes were observed in 
30 minutes. Irradiation doses less than 2,000 r induced no morphologic 
changes in the cerebellum during the observation time ot 6 months. 

A few typical experiments will indicate the nature, se\erity, and sequence 
of damage after exposure of the cerebellum of mice to different x-ray doses. 

ToT.\L Necrosis of Cerebellar Tissue Following Exposure to X-rav 
Doses Ranging from 60,000 to 10,000 r 

In the range from 60,000 to 10,000 r. x-irradiation induced total necrosis 
of cerebellar tissue within a time depending on the dosage. 

191 



192 NORBERT SCHUMMELFEDER 

In 30 minutes following exposure to 60,000 r, swelling of nuclei and 
cytoplasm occurred in granule cells, Purkinje cells, and basket cells in deeper 
parts of the molecular layer. The interstitium of the molecular layer exhib- 
ited slight vacuolation. 

In 1 hour after 60,000 r, there was greater swelling of the various nerve 
cells, and the granular layer was loosened. Vacuolation of the molecular 
layer was increased. The chromatin in the swollen nuclei of the granule 
cells was condensed in the form of irregularly shaped coarse bodies located 
on the nuclear membrane. The swollen nerve cells within the molecular 
layer had a clear space around their nuclei. These spaces contained small, 
thread-like, ragged or flaky, cytoplasmic residues, often attached to the 
nuclei (acute cell swelling). The degree of nerve cell swelling was slight in 
upp)er parts of the molecular layer and increased progressively down to the 
Purkinje cells. Within the molecular layer, the nuclei of glial cells were 
slightly swollen or occasionally pyknotic. Vacuolation (status spongiosus) 
of the molecular layer was evident ; the vacuoles were at first round, then 
oval, and increased in size downward from the cerebellar surface to the 
Purkinje cell layer. Occasionally, the vacuoles were arranged in vertical 
columns. The vacuoles seemed to contain a protein-free aqueous solution, 
because even with special staining methods and histochemical reactions no 
other material could be demonstrated. Henceforth, the Purkinje cells, in 
particular their nuclei, showed hydropic swelling. Within the swollen cyto- 
plasm the Nissl bodies had usually disappeared. In other cells, the NissI 
substance was dispersed as dust-like particles over the entire cytoplasm. The 
Bergmann glial cells occasionally had swollen nuclei, but they seldom dis- 
played any nuclear pyknosis. 

At 2 hours after 60,000 r, the regressive changes were still more advanced 
(Figs. 1 and 2). Within the lower part of the molecular layer, the vacuola- 
tion had progressed to tissue sponginess. Within the uppermost parts of the 
granular layer, the tissue looseness had also increased. Some nuclei of the 
irradiated granule cells were no longer swollen, but were shrunken and 
pyknotic. Correlated with the decrease of the x-ray dose with distance 
traversed, there was an upper zone composed mostly of pyknotic nuclei, 
then a transitional zone with pyknotic as well as swollen nuclei, and a 
lower zone containing solely swollen nuclei. Apparently as a consequence 
of the pressure from the swelling of the molecular and granular layers, 
many Purkinje cells within the center of the damaged area were deformed. 
These cells were oval, and their longitudinal axes were parallel to the gran- 
ular layer. In the lateral part of the irradiated field, several Purkinje cells 
exhibited acute swelling. Near the border of the damaged area, which was 
evident because of the changes in the granular layer, the Purkinje cells 
showed slight or no morphologic alterations. 











7^ 









Figs. 1 and 2. Radiation damage 2 hours after exposure to 60,000 r. Plane of the 
section is parallel to the direction of the x-ray beam. Vacuolation of molecular layer, 
shrinkage of Purkinje cells, and pyknosis of the nuclei of granule cells occur in the 
most severely damaged area of the granular layer, and nuclear swelling in the lower 
part. Fig. 1 : X 40. Hematoxylin-eosin. Fig. 2: X 170. v. Gieson-stain. 

193 



194 



NORBERT SCHUMMELFEDER 



At 4 to 5 hours after x-irradiation with 60.000 r. nearly all the granule 
cells in the irradiated part of the granular layer were shrunken and their 
nuclei pyknotic (Fig. 3). These cells were surrounded by a clear space. 
In the molecular layer, the nuclei of the swollen nerve cells were now 
pyknotic, while the degree of vacuolation in this part of the irradiated 
cerebellar cortex was the same as before. The morphologic changes in the 
molecular and granular layers were strictly localized, limited to the irradi- 
ated field. The border betw-een the irradiated and the unchanged cere- 
bellar tissue was sharp, as though drawn with a ruler. During this time 
interval, the white matter of the cerebellum was slightly, if at all, modified 
by the irradiation. There was neither detectable edema in the white matter 
nor any substantial change in the axis cylinders or in glial cells. 

Within the first hours after irradiation, no e\idence of a conspicuous 




Fig. 3. At 4 hours after irradiation with 60,000 r, showing severe vacuolation of 
the molecular layer, shrinkage of Purkinje cells, and pyknosis of granule ceil nuclei. 
X 780. Hematoxylin-eosin. 



SEQIENCE OF X-RAY DAMAGE IN MOUSE CEREBELLUM 



195 



change in the blood vessels was observed. Occasionally a slight dilatation 
of the capillaries was seen as an expression of hyperemia. 

In 5 to 6 hovns after x-irradiation of a 3 X 3 mm field with 60,000 r 
the mice died. Further evolution of radiation damage could be followed 
only after use of a dose of 60,000 r through a smaller aperture 0.5 X 2 mm) 
or by using lower x-ray doses (40,000 to 20,000 ri. 

At 12 to 14 hours after 60.000 r. using a 0.5 X 2 mm field, the pyknotic 
nuclei of the granular layer underwent disintegration, mostly as karyorrhexis. 
The damaged area was still limited to the irradiated field, as a section cut 
transversely to the direction of the x-ray beam illustrates ( Fig. 4 ) . The 
Purkinje cells also exhibited signs of disintegration. Some of these cells had 
greatly swollen cytoplasm and nuclei and showed lytic changes ( Fig. 5 ) . 
Within other Purkinje cells, the nuclear chromatin was initially condensed, 
simulating pyknosis, and then the cytoplasm and nucleus underwent lysis. 

At 20 to 30 hours after irradiation with 60.000 to 40.000 r. necrosis was 
completely established in the superficial part of the cerebellar folia, i.e.. 
nearest the radiation soiuce ( Fis.. 6 ) . The necrosis was strictly limited to 








'J:rM - 







Fig. 4. Radiation damage 12 hours after exposure to 60.000 r (0.5 X 2 mm field). 
Plane of section is trans\erse to direction of the x-ray beam. Pyknosis and disinte- 
gration of nuclei in the granular layer. X 4(1. Hematoxylin-eosin. 




Fig. 5. Different types of nerve cell change after irradiation: (a) condensation of 
the nuclear chromatin, (b) swelling and partial vacuolation of the cytoplasm, (c) 
severe swelling of the cytoplasm, (d) severe nuclear and cytoplasmic swelling, in- 
cipient lysis of the cytoplasm. X 1440. Gallocyanin-chromalum stain. 



196 



SEQUENCE OF X-RAY DAMAGE IN MOUSE CEREBELLUM 197 




198 NORBERT SCHUMMELFEDER 







i 



Hi • 1^* .^•^^ • H T •:] 

Fig. 6. At L!U hours alter irradiation with lU.OOU r, showing the honiogenization type 
of nerve cell necrosis. X 700. Hematoxylin-eosin. 



the irradiated field and was sharply demarcated from the nonirradiated part 
of the cerebellum. The molecidar layer showed granidar and clumped areas 
of disintegration. Only some of the nerve and glial cells were preserved. 
Most of them contained pyknotic nuclei and showed all stages of disinte- 
gration or lysis. Within the necrotic granular layer enlarged by edema, the 
preserved nuclei were pyknotic, and between them nuclear debris was 
frequently found. Hemorrhages and extravasations of plasma proteins were 
remarkable neither within the necrotic granidar layer nor within the white 
matter. The nuclei of the glial cells in the white matter of the more super- 
ficial part of the cerebellar folia (nearest the radiation source) were pyk- 
notic, and in deeper parts of the cerebellum they were swollen. Some blood 
vessels in the necrotic area were preserved, but dilated. Frequently, they 
were surroimded by a hollow space, the ground membrane had often under- 
gone hyaline thickening (hyalinosis) , and there was swelling of endo- 
thelial and adventitial cells. 

Within the irradiated field the Purkinje cells had, in part, disappeared. 
Most of the preserved Purkinje cells showed homogenization. They con- 
tained pyknotic nuclei and exhibited a strong cytoplasmic eosinophilia. 
Many were in all stages of disintegration, including cell shadows. 

At 90 hours after irradiation^ necrosis was completely established in all 



SEQUENCE OF X-RAY DAMAGE IN MOUSE CEREBELLUM 



199 



parts of the irradiated cerebellar tissue (Fig. 7). Within the necrotic area 
there were sca\enger cells and gitter cells as well as proliferating and 
naked glial cells. The nuclei of most altered granule cells were disintegrated 
and lysed. Only segregated groups of pyknotic nuclei together with nuclear 
debris were observed within the irradiated field. In contrast to the animals 
of shorter survival, edema was seen within the Bergmann layer and in 
adjacent parts of the molecular and granular layers. Frequently, the 
edematous process extended a short distance into the nonirradiated part 
of the Bergmann layer and was associated with hydropic swelling of adjacent 
Purkinje cells. Homogenization of Pinkinje cells was no longer found, as 
cells which had suffered this change seemed to have been removed. 

When the survival period was extended by reducing the x-ray doses to 
20,000 to 10,000 r, resorption and repair occurred in the irradiated cere- 
bellar tissue in the same manner as in necrosis of brain tissue resulting from 
other causes. The final stage consisted of cystic licjuefaction of the necrotic 
brain tissue. By 20 days after irradiation with 16.000 r much of the necrotic 
cerebellar tissue had been removed. The resulting cyst-like areas contained 
remnants of necrotic debris and were traversed by partly preserved blood 
vessels. The processes of resorption and repair were e\ident at the margin 




^ri; 








Fig. 7. Total tissue necrosis 90 hours after irradiation with 70.000 r (0.5 X 2 mm 
field). X 35. Hematoxylin-eosin. 



200 NORBERT SCHUMMELFEDER 

of the damaged brain tissue in the form of numerous gitter cells. Within 
the preserved tissue only slight glial reaction had occurred. 

Partial Necrosis of Cerebellar Tissue Following Exposure to X-ray 
Doses from 10,000 to 4,000 r 

At radiation doses from 10,000 r down to 4,000 r, there was necrosis only 
of the granular layer and a loss of single nerve cells in the other parts of 
the cerebellar cortex. The latent period between irradiation and the develop- 
ment of morphologic changes was longer than with higher doses. 

Thus, with a dose of 5,000 to 4,000 r, the first morphologic changes 
appeared at approximately 12 hours. At this time only a few scattered 
pyknotic nuclei of granular cells were observed. The other parts of the 
irradiated cerebellar tissue were not modified. 

At 5 days the more superficial part of the granular layer had undergone 
partial dissolution. Only adjacent to the Purkinje cell layer was there a 
zone of preserved pyknotic granule cell nuclei, and it was narrow. Occupy- 
ing regions in which granule cells had undergone dissolution were numerous 
gitter cells and i.solated pigment-bearing scavenger cells. Purkinje cells were 
destroyed only in the most severely damaged regions of the irradiated field. 
In their place, proliferated glia were seen. Other Purkinje cells were being 
phagocytized. The cerebellar white matter was loosened strikingly, and in 
some foci the white matter was completely destroyed. Some of the blood 
vessels, especially the capillaries, were greatly dilated and were surrounded 
by a mantle of mononuclear cells including a few neutrophilic leucocytes. 
Endothelial cells of occasional blood vessels were swollen. 

At 10 days after irradiation the destroyed Purkinje cells were replaced 
by glial shrubbeiies ( Gliastrauchwerk) extending from the Bergmann layer 
into the molecular layer. The uppermost part of the molecular layer showed 
decided shrinkage, as did other parts of the cortex (Fig. 8). 

At 20 days after exposure to 5,000 r, extensive perivascular hen:iorrhages 
were often found in damaged cerebellar tissue. Shrinkage, pronounced glial 
proliferation, and cicatrization (gliosis) occurred in the irradiated tissue. 
The glial fibers within the damaged molecular layer, particularly those of 
the Bergmann cells, were thickened remarkably. These coarse fibers could 
easily be demonstrated by Lendrum's (1947) method. 

Persisting Piukinje cells and nerve cells of the inolecular layer often 
showed regressive changes, e.g., swelling of cytoplasin and nucleus or 
chromatolysis. Even at this time interval, some of the granule cells in the 
irradiated area were pyknotic. Such pyknosis had probably developed in 
the course of the radiation damage. The periphery of the most severely 
damaged area was marked off by pronounced vascularisation. The vessels 



SEQUENCE OF X-RAY DAMAGE IN MOUSE CEREBELLUM 201 








Fig. 8. Radiation damage ID days after irradiation witli 5.01)0 r. Dissolution of 
central parts of the granular layer and destruction of Purkinje cells within the upper- 
most layer followed by glial proliferation extending into the shrimken molecular layer. 
X 215. Gallocvanin-chronialiun. 



were irreoiilaily dilated, and their walls showed remarkable hyalinosis. 
Circumscribed aneurysmal distention of smaller blood vessels, previously 
described by Scholz ( 1934a, b. 1937) in brain tisstie damaged by irradiation, 
was noted at the 20-day sta^e. 

At 40 days after irradiation, the ylial reaction in the irradiated cerebellar 
tisstie had progressed further. The entire damaged area was shrimken. Some 
sjranule cells were preserved, but it was difficidt at times to distinguish 
them from proliferated glial cells. As at shorter time intervals, hemosiderin- 
containing sca\enger cells were often seen. Hemosiderin indicated previous 
hemorrhage. 

In contrast to higher x-ray doses, 5,000 to 4,000 r produced only partial 
necrosis of the cerebellar tissue. The molecular layer and the white matter 
of the cerebellar folia were little affected. All the well known processes of 
resorption and repair took place in the irradiated cerebellar tissue in the 
same manner as in partial cerebellar necrosis due to other cause. Glial 
scarring was the final outcome. 



202 NORBERT SCHUMMELFEDER 

Single Cell Necrosis Within the Cerebellum After Exposure to 
X-RAv Doses Below 4,000 r 

X-ray doses below 4,000 r resulted in loss only of single cells or small cell 
groups, not in necrosis of larger areas of the irradiated cerebellar tissue. 
The latent period between irradiation and the appearance of damage was 
increased. Following an x-ray dose of 3,000 r, morphologic changes within 
the irradiated cerebellar tissue were not observed in less than 45 days. At 
this time, focal loss of granule cells and destruction of some Purkinje cells 
were noted in the most superficial cerebellar folia, i.e., those nearest the 
radiation source. The better preserved Purkinje cells often showed regressive 
changes, which were of differing types. Cell shadows were occasionally 
observed. Together with fresh hemorrhages within the damaged brain tissue 
were residues of older ones in the form of hemosiderin in scavenger cells. 
The blood vessels were dilated, and their walls often showed hyalinosis. 
Perivascular infiltrates of mononuclear cells were found over the entire 
irradiated field. 

At 4 months after irradiation with 3,000 r the various components of the 
irradiated cerebellar folia were relatively well preserved. The granular layer 
showed patchy looseness due to disseminated loss of granule cells. This layer 
contained proliferated glial elements and was traversed by capillaries. 
Hyaline thickening was found in the wall of blood vessels in the lepto- 
meninges and brain tissue. The lumens of many vessels were dilated. Some 
Purkinje cells were destroyed and replaced by glial nodules from which 
glial shrubberies extended into the molecular layer. The preserved Purkinje 
cells seemed imaltered. 

Early Changes in Cellular Nucleoproteins of Irradiated 
Cerebellum 

All these observations indicate that local irradiation of the cerebellum of 
mice with sufficiently high doses of x-rays results in marked alterations of 
cerebellar tissue which are limited to the irradiated field. One of the most 
remarkable changes is the pyknosis of nuclei of granule cells. Another im- 
portant observation is that during the course of the radiation damage dif- 
ferent types of regressive changes are observed in the Piukinje cells. 

We have applied the fluorescence technique, using the basic fluorochrome 
acridine orange, to study alterations in nerve cells. This histochemical 
method is especially suitable for investigation of cytoplasmic and nuclear 
nucleic acids (Schiimmelfeder et al., 1958). Using buffered acridine orange 
solutions, pH 4.0 to 7.0, all ribonucleic acid (RNA ) -containing material, 
e.g., the cytoplasm of Purkinje cells, fluoresces bright orange or red. In 



SEQUENCE OF X-RAY DAMAGE IN MOUSE CEREBELLUM 203 

contrast, the highly polymerized deoxyribonucleic acid (DNA) of the 
nuclei shows an intense yellow or yellow-green fluorescence. Other elements 
of the brain tissue present a weak green fluorescence. The diff"erence in the 
staining raction of RNA and DNA is due to the high degree of polymeriza- 
tion of the nuclear DNA in comparison with the cytoplasmic RNA. After 
depolymerization, e.g., by placing the tissue slices in boiling water or 
hydrochloric or perchloric acid, the DNA stained with acridine orange 
shows a red fluorescence. The depolymerized DNA behaves, therefore, like 
RNA, which is always more weakly polymerized. The acridine orange 
method is more sensitive than the methyl green, which can, however, bring 
out wide differences in the degree of polymerization in DNA. The occur- 
I'ence of depolymerization in DNA thus can be proved by using the acridine 
orange fluorescence technique. 

We tried to determine with this method whether alterations occur in the 
structural organization of the DNA in the pyknotic nuclei of irradiated 
granule cells. Many other cells besides those of the cerebellar granular layer 
show clumping or other changes in the nuclear chromatin. In an histo- 
chemical study of nuclear changes in the superficial epithelium of the 
tongue of mice that had received radioactive chromic phosphate, Burstone 
(1953) observed enlargement and hyperchromasia of the nuclei. In contrast 
to nonirradiated controls, treatment with deoxyribonuclease (DNase) re- 
sulted in decreased staining capacity of these nuclei to the Feulgen reaction 
and to the methyl-green-pyronin method. Burstone (1953) believed that 
this decreased resistance of the irradiated nuclei is due to a somewhat 
decreased aggregation of the nuclear DNA. Based on such observations and 
on radiation experiments on DNA solutions which show a splitting of DNA 
molecules i Scholcs and Weiss, 1952) some still believe that the clumping 
of chromatin after irradiation is a result of depolymerization. If such 
depolymerization of DNA should occm- in the pyknotic nuclei of irradiated 
cerebellar granule cells as a primary effect or a secondary reaction to ioniz- 
ing radiation, then it should be demonstrable with acridine orange by the 
nuclei exhibiting red fluorescence. We ha\e ne\er found this. Disregarding 
the point that pyknosis causes a higher density which provokes a somewhat 
more intense fluorescence, no difference in the fluorescent color of these 
pyknotic nuclei compared to normal nuclei has been obser\ed. 

The fact that the DNA of these pyknotic nuclei is not significantly 
depolymerized can also be shown by using methyl green. This dye stains 
only highly polymerized DNA (Kurnik. 1950. 1952; Kurnik and Forster, 
1950: Kurnik and Mirsky, 1950; Pollister and Leuchtenberger, 1949; Ver- 
cauteren. 1950). The pyknotic nuclei should not be stainable with methyl 
green if there had been depolymerization of nuclear DNA. But, as compared 
with nonirradiated cells, no substantial diflference in the staining capacity 



204 NORBERT SCHCMMELFEDER 

of the pyknotic nuclei was observed. This result corresponds to that of 
Sparrow et al. (1952) on Trillium nuclei. 

Analogous to Burstone's (1953) observations, Kaufmann et al. (1955) 
have shown in experiments on meristematic cells of onion roots that DNA 
in irradiated cells is more easily dissoKed by DNase than in nonirradiated 
controls. In contrast, similar experiments on grasshopper embryos showed a 
higher resistance of the irradiated nuclei, i.e., by their DNA. to enzymatic 
hydrolysis. These different results stimulated us to seek information on 
whether the de\elopment of pyknosis of granule cell nuclei following ir- 
radiation alters their response to depolymerizing and hydrolyzing agents. 

The results of these experiments showed that pyknotic nuclei of granule 
cells are more resistant than nonpyknotic nuclei to treatment with depoly- 
merizing agents, e.g. to boiling water or to hydrochloric or perchloric acid. 
Pro\ided the reaction conditions are favorable, only in the nonirradiated 
nuclei did exposure to these agents result in depolymerization. These nuclei 
showed red fluorescence after staining with buffered solutions of acridine 
orange, pH 5.0 to 7.0, whereas the pyknotic nuclei still fluoresced bright 
yellow owing to retained high polymerization of DNA. The difference in 
color and intensity ot the fluorescence was so conspicuous that each indi- 
vidual pyknotic nucleus could easily be obser\ed. 

Methyl green, which stains only highly polymerized DNA, has yielded 
the same results in companion sections. The pyknotic nuclei were still stain- 
able with methyl green, whereas unaltered granule cell nuclei could not 
be stained after pretreatment with depolymerizing agents. 

Further experiments showed that enzymatic breakdown of DNA, using 
DNase, occurs more slowly in pyknotic than in intact nuclei. This result is 
similar to that ob.served by Kaufmann ct al. (1955) in irradiated grass- 
hopper embryos. 

Hydrolysis with hydrochloric or perchloric acid removed DNA from the 
nuclei of unaltered granule cells, whereas DNA of the pyknotic nuclei in 
irradiated granule cells was only slightly depolymerized. Using favorable 
conditions of hydrolyzation. it is easy to demonstrate selectively the pyk- 
notic nuclei after staining with acridine orange, whereas nonpyknotic nuclei, 
which are not altered by the irradiation and which are deprived of the 
DNA by the foregoing hydrolysis, remain imstained. 

In our experimental study we were unable to determine whether increased 
resistance of the irradiated pyknotic nuclei to DNase and to depolymerizing 
and hydrolysing chemical agents is an immediate and specific effect due to 
the action of ionizing radiation on the DNA of the nuclei. The observations 
of Kaufmann ct al. (1955) have indicated that the structural organization 
of the nucleoproteins in irradiated nuclei is changed. On the other hand 
Yakar (1952) has demonstrated in plant cells that the speed of enzymatic 



SEQUENCE OF X-RAY DAMAGE IN MOUSE CEREBELLUM 205 

hydrolysis of chromatin decreases if pyknosis is induced by chemical asents. 
It is conceivable that the increase in resistance of the pyknotic nuclei which 
we found is attributable to greater density of the nuclear mass in that the 
increased density reduces depolymerization and hydrolysis. 

We have emphasized that when RNA-containinsi material, e.g., the cyto- 
plasm of the Purkinje cells, is stained with acridine oranse. pH 4.0 to 7.0, 
it takes on a bright orange or red fluorescence. Since different types of 
regressive changes can be observed in Purkinje cells during the course of 
radiation damage, we have used the acridine orange method to study the 
behavior of their cytoplasmic nucleic acids. Because of regressive changes 
in these cells and because of the nuclear pyknosis in granule cells, the irradi- 
ated area of the cerebellar tissue can easily be demonstrated by this method. 
Since regressive changes in the Purkinje cells usually occur more strikingly 
in the center of the irradiated area than along its margins, the red fluo- 
rescence exhibited by the more peripheral cells gradually decreases in in- 
tensity toward the center of the irradiated zone. The cytoplasm of unaltered 
Purkinje cells fluoresces bright orange-red. Acute shrunken nei-ve cells show 
the same fluorescence because their cytoplasm contains abimdant RNA. 
Immediately following" irradiation, the swollen and \acuolated Purkinje cells 
give off a slightly decreased orange fluorescence, but after sufficient time 
has passed the cytoplasm exhibits only yellow or yellow-green fluorescence 
because the RNA content of their altered cytoplasm is decreased. Purkinje 
cells showing the homogenization type of necrosis have a green fluorescence 
because they have lost all cytoplasmic RNA. Since color and intensity of 
fluorescence in these Purkinje cells is similar to that of the neuroglia of the 
molecular layer, it is somewhat difficult to recognize necrotic and homog- 
enated Purkinje cells. It bears emphasis that the nuclear pyknosis in Purkinje 
cells as well as in granule cells is not associated with depolvmerization of 
DNA, since, when stained with acridine orange, the pyknotic nuclei still 
fluoresce yellow-green. 

Discussion 

There ha\e been apparently conflicting reports in the literature as to the 
primacy of irradiation damage in the central nervous system, whether in 
vessels or in nerve cells. The cerebellum seems especially suitable for in\esti- 
gation of this problem. .Some other workers already ha\e directed attention 
to the radio\ulnerability of this part of the brain. In macaque monkeys, Hay- 
maker et al. ( 1958) ha\-e studied the effect on the central nervous system of 
whole-body BA'^"-LA"" i gamma i radiation. E\ idence of ner\e cell damage 
in the cerebrum was scanty, but granule cells of the cerebellum were pyknotic 
within a dose range of 5,000 to 30,000 r. The pyknosis occurred earlier and 



206 NORBERT SCHUMMELFEDER 

was more severe at higher dose levels. In other experiments on the macaque, in 
which 10,000 r Co*'° (gamma) radiation to the head alone or to the whole 
body was used, Vogel et al. (1958) also observed pyknosis of granule cells 
in the cerebellum. Both Haymaker et al. (1958) and Vogel et al. (1958), 
whose animals survived no longer than one week, observed that the pyknosis 
of granule cells was reversible by about the 3rd day. In a subsequent study 
of the effects of cobalt'"^ (gamma) radiation on the cerebellum of macaque 
monkeys at the same dose levels, Wilson (1960) confirmed the observation 
that under these conditions the granule cell pyknosis is transitory and 
reversible, but he found that pyknosis was somewhat briefer than reported 
by the other authors. Similar results have been obtained in rabbits after 
exposed to a Co"" source (Vogel, 1959) and in guinea pigs after x-irradiation 
(Alvord and Brace, 1957). Vogel (1959) noted that after a dose of 15,000 r 
gamma radiation from a Co''" source, granule cell pyknosis was evident in 
15 hours and that by the 10th day practically all the granule cells of exposed 
folia had disappeared. According to Hicks (1953; Hicks and Montgomery, 
1952; Hicks and Wright, 1954; Hicks et al.. 1956) the same holds for rats 
and mice. In these animals they found that nerve cells or cerebellar tissue 
could readily be damaged by x-rays, depending on the dose. 

Our observations coincide with those of Hicks et al. { 1956) on the mouse. 
We have shown that sufficiently intense x-irradiation of the cerebellar tissue 
results in primary tissue changes. Incidence, pattern, and course of these 
changes are clearly related to the x-ray dose. Under the conditions of oiu' 
experiments, nuclear pyknosis in irradiated granule cells of adult mice is a 
sign of irreversible cellular change leading to cellular necrosis. In this respect 
our observations on mice do not coincide with those of Haymaker et al. 
(1958), Vogel et al. (1958), and Wilson (1960) on macaque monkeys. We 
are imablc to explain this difTerence, but points to be taken into considera- 
tion are species and age of the animals, nature of irradiation, radiation 
energy and rate of dosage. 

There is still little knowledge of the earliest histopathologic and histo- 
chemical changes occurring in the cerebellum following irradiation or in the 
sequence in which the changes develop. Our histochemical investigations 
show that during early radiation damage of cerebellar tissue following high 
x-irradiation dose, changes occur in the nucleic-acid-containing components 
of granule and Purkinje cells. Particularly in Purkinje cells, alterations occur 
in the cytoplasmic RNA that are secondary eflfects due to regressive cellular 
alterations, since swelling of the nerve cells was observed initially and de- 
crease in cytoplasmic nucleic acid content later on. The change in the 
structural organization of the nuclear DNA in the pyknotic granular cells is 
possibly also a secondary effect due to the increased density of the pyknotic 
nuclei. But it could also represent a primary change in the physicochemical 



SEQUENCE OF X-RAY DAMAGE IN MOUSE CEREBELLUM 207 

quality of the DNA caused by the action of ionizino radiation. Further 
in\estigations are necessary to clarify this problem. As brought out by the 
acridine orange fluorescence technique, the increased resistance of pyknotic 
nuclei to depolymerizing and hydrolysing agents allow clear identification of 
damaged irradiated pyknotic cell nuclei. 

Summary 

Dexelopment and course of radiation lesions in the cerebellum in mice 
were studied after exposure to single doses of x-rays, 250 to 60,000 r, applied 
to one cerebellar hemisphere through apertures 3 X ^ mm or 0.5 X 2 mm 
in diameter. Degree and sexerity of radiation damage in the cerebellum was 
correlated in terms of tinre-intensity relationships with the x-ray dose. 

After a dose of 60.000 r. morphologic changes of different cerebellar struc- 
tures (e.g.. nerve cells of the molecidar layer and Purkinje and granule cells) 
were visible as early as 30 minutes following exposine and were fully dcxel- 
oped at 60 minutes. Within 90 hours, complete necrosis of the irradiated 
cerebellar area with concomitant resorptive and reparative changes was 
observed. C'ystic liquefaction eventually occurred. 

After less intense x-ray doses, 60,000 to 10,000 r. similar radiation lesions 
were observed, but the latent period of their inception was longer. Exposure 
to doses below 10,000 r down to 4,000 r residted in necrosis of the granidar 
layer and in destruction of single nene cells in other parts of the cerebellar 
cortex. The latent time was more prolonged. Radiation damage finally resulted 
in the formation of a glial scar. 

At x-ray doses below 4,000 r. only single nerve cells or cell groups were 
damaged, and the latent period was correspondingly lengthened. In such 
lesions, proliferation of glial elements and capillaries ultimately occurred, 
and hyalinosis developed in the walls of distended blood vessels. Since no evi- 
dence of vascular alterations was observed before development of nerve cell 
lesions, the visible radiation damage was interpreted as due to a direct effect 
of the ionizing radiation on the cellular elements of cerebellar tissue. 

The acridine orange fluorescence tcchniciue was used to determine whether 
alterations occin- in the structural organization of the DNA in pyknotic 
nuclei of irradiated granule cells and in the RNA content of Purkinje cells. 
Our results indicated that after irradiation DNA of pyknotic nuclei in the 
granular layer is not depolymerized to a noticeable degree. These pyknotic 
nuclei were more resistant than normal to treatment with depolymerizing 
and hydrolysing chemical agents and DNase. Without fiuther experimenta- 
tion it is difficult to say whether this increased resistance of irradiated, 
pyknotic nuclei is an immediate and specific effect due to the action of ioniz- 
ing radiation on the DNA or whether it is a nonspecific effect due to in- 



208 NORBERT SCHCMMELFEDER 

creased density of the nuclear mass. Within the region damaged by irradia- 
tion, Purkinje cells in a state of regression showed remarkable decrease or 
loss of cytoplasmic RNA which was obviously secondary to cellular 
regression. 

Acknowledgment 

The author wishes to acknowledge, with appreciation, the assistance 
rendered in these studies by Dr. Bergeder and Dr. Ebschner, University of 
Bonn, Germany, and the late Dr. E. Krogh, University of Aarhus, Denmark. 



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210 NORBERT SCHUMMELFEDER 

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Morphological Effect of Repeated Low 
Dosage and Single High Dosage Application 
of X-lrradiation to the Central Nervous 

System * 

WiLLIBALD SCHOLZ. WoLFGANG ScHLOTE. AND WOLFGANG HiRSCHBERGER 

Deutsche Forschungsanstalt fur Psychiatrie 
Max-Planck-Institut, Munich, Germany 



This preliminary presentation deals with the effects of x-rays on the tissue 
of the central ner\oiis system and the ensuinij pathogenesis. Although we 
shall refer repeatedly to x-ray dosage, its relation to the time of manifestation 
of tissue changes, and the se\erity of these changes, it is not our intention 
to determine an exact time-dose relationship as this has been done by Berg 
and Lindgren (1958). We ha\e tried to find a connection between the 
delayed x-ray lesions seen after moderate single or fractionated doses and 
the acute tissue necroses occurring within hours after a single application of 
massi\e doses of hiyh intensity up to 80.000 r. It is now almost universally 
accepted that the delayed lesions originate from changes ot the vessels. A 
breakdown of the hematoencephalic barrier has been considered significant 
since Mogilnitzky and Podljaschuk ( 1930) described the passage of trypan 
blue into repeatedly irradiated central nervous tissue. When we made our 
first investigations and experiments with dogs in 1932-1935 (Lyman et al., 
1933; Scholz. 1935), the histologic picture was dominated to such a degree 
by plasmatic transudations with and without erythrodiapedesis into the 
central nervous tissue, it was difficult to admit any direct influence of the 
x-rays on ner\ous tissue constituents. While it was true that the x-ray doses 
applied through different portals to the skull ranged from 4,400 r to about 
8.000 r, the intensity of irradiation was so low that to apply 12 skin erythema 
doses corresponding to about 6.600 r. a radiation time of 6 hours was 
required. Since that time, x-ray technicjue and the accurate measurement of 
dosage has improved so considerably that it seemed worthwhile to re-examine 
oiu" former results with new experimental material and methods. 

* This project was supported by the School of .\\iation Medicine of the .^ir Re- 
search and Development Command. U.S. An Force, through its European Office. 

211 



212 W. SCHOLZ, W. SCHLOTE AND W. HIRSCHBERGER 

Accordinoly, since 1956, we have studied the neuropathology of x-ray 
lesions produced by irradiation of the spinal cord of rabbits.^ A 3 x 6 cm 
field on the back, corresponding to the upper thoracic segments of the cord, 
was irradiated. The technical conditions were: 180-200 kv, 18 mA, 0.95- 
1.12. Cu half value layer, filter 0.5 Cu. 60 r per min, focus — skin distance 
50 cm, and pendulum angle 70° on both sides. The average dose was 250 r 
daily; total doses were 3,000 to 1 1,000 r given over 12 to 40 days. All animals 
acquired paralysis of the hind legs and loss of sphincter function within 4 to 
33 weeks after the beginning of irradiation. This investigation was published 
in Psychiatria ct Nciirologia Japonica, 1959, and only the important features 
will be mentioned here. The white matter of the spinal cord was much 
more affected than the gray substance and showed more or less circum- 
scribed areas of disintegration following the radial distribution of the spinal 
vessels entering from the vasocorona along the whole periphery of the cord. 
Regressive changes of the small vessels with slight perivascular astrocytic 
reaction within the focal lesions suggest the transudation of a fluid histo- 
logically not demonstrable, causing swelling and disintegration of the myelin 
fibers. Only in a few cases did the gray substance participate in the changes. 
Here, plasmatic extravasation, partly with erythrodiapedesis, could be ob- 
served, followed by an astrocytic reaction and some regressive changes in 
some nerve cells. Although a reaction of the neuroglia was observed, it 
remained rather scanty, especially in the white matter. Regardless of the 
fact that some lesions were 3 to 4 weeks old, sudanophilic material was not 
observed. In both white and gray matter, plasmatic disintegration of the 
walls of larger vessels was encoimtered occasionally, but plasmatic exudation 
was seen only in the gray substance. Thus the morphologic feature patho- 
genetically pointed to the primacy of processes of transudation, exudation, 
and erythrodiapedesis, that is, to a breakdown of the barrier function of the 
spinal vessels. This concept was further supported by intravital injection of 
tiypan blue (Fig. 1). There are blue stained foci in the white matter 




Fig. 1. Delayed lesion of the spinal cord of a rabbit after fractionated x-irradiation, 
intravitally stained with trypan blue. Beneath some foci of disintegration in the white 
matter, the whole gray substance although demonstrating no disintegration, has taken 
the blue color. 

' We received the material from the radiologist Dr. Breit, who was interested in 
questions of tolerance dose, dose fractioning, and concentric application by a pendulum 
x-ray machine. 



MORPHOLOGICAL EFFECT OF X-RAYS TO THE CNS 



213 



caused by destruction of tissue. The remarkable fact is the sharply limited 
blue stainino' of the whole gray matter, which demonstrates no disintegration 
at all. 

These investigations have been continued in 21 additional adult rabbits. 
Equivalent changes in the spinal cord could be produced using the same 
technique with fractionated doses totaling 3,000 r. No exact relation between 
the amount of applied r and the length of the interval can be stated, but on 
the average the intervals became longer with diminution of the total dose. 
The minimum single dose sufficient to produce severe changes in the cord 
after 5 months was 2.000 r. Some figines demonstrating the pathologic 
findings in this material show again the important role which the breakdown 
of the blood-brain barrier plays in the pathogenesis of changes in the 
nervous tissue. Thus we see in Fig. 2 that the numerous focal changes in 
the white matter follow the distribution of the small arteries entering the 
cord from the vasocorona. In this case, a single dose of 3,500 r was followed 
by paralysis 15 weeks later. Two small vessels in the white matter (Fig. 3) 




Fig. 2. Delayed lesion of the spinal cord of a rabbit, 3% months after a local 
single x-ray dose of 3,500 r. Numerous areas of disintegration in the white matter fol- 
low the radial direction of the entering vessels of the vasocorona. Myelin stain 
(Schroder). 



show a swelling and disintegration of their walls cau.sed by infiltration with 
a plasmatic material which is stained yellow in van Gieson preparations. 



214 



W. SCHOLZ, W. SCHLOTE AND W. HIRSCHBERGER 




Fig. 3. Plasmatic swelling and disorganization of the walls of two small \essels in 
the white matter, still without effect on the neighboring tissue, 5 months after a single 
dose of 2,000 r. van Gieson. 



No change of the neighboring myelin fibers can be demonstrated. These 
changes occurred 5 months after the application of a single x-ray dose of 
2,000 r. Within an area of spongy dissolution of the white matter in the 
same case, plasmatic material with red blood corpuscles spreads out from 
such vessels (Fig. 4). These conditions are demonstrated more distinctly 
with the Mallory method in Fig. 5. The fibrinoid disorganization of the 
vessel walls is here followed by erythrodiapedesis, hemorrhages, and fibrin- 
containing fluid in the gray matter. In all places where the plasmatic fluid 
spreads into the tissue, oxygen diff"usion is inhibited and the cellular ele- 
ments become necrotic. 

On the whole, the findings in this second series confirm and complete the 
results of our first investigation on x-ray changes in the spinal cord of adult 
rabbits. The pathogenic mechanism seems the same as in the brains of 
dogs, observed more than 20 years ago. We have not seen any proof for a 
primary effect of ionizing radiation on the neuronal constituents of the tissue. 
Whenever a damage of neuronal constituents could be observed, it was 
accompanied and often preceded by changes in the barrier function. The 
focal lesions of the white matter are distributed irregularly within the field 
of x-irradiation of the cord. Some sections are filled with areas of demyelina- 
tion, whereas at other levels not a single one can be seen. This may account 



MORPHOLOGICAL EFFECT OF X-RAYS TO THE CNS 215 




Fig. 4. Plasmatic mhltration and erythrodiapedesis into the tissue in an area of 
disintegration of the white matter from the same case as Fig. 3. van Gieson. 



for the fact that in routine in\estiiiations of single cases, no morphologic 
changes may be observed, although the animals are paralyzed. 

In the second series, transformation of the tissue debris into sudanophilic 
material could not be observed, although in many cases the clinical symp- 
toms indicated that morpholo2,ic changes were 3 to 4 weeks old. 

In addition to these experiments on the spinal cord of rabbits, the brains 
of approximately 100 Syrian hamsters were irradiated to examine the effect 
of a high and intensi\e single x-ray dose using a special Siemens x-ray 
machine with a beryllium tube. The animals were fixed on a small table 
(Fig. 6 I, and the whole body was covered v,ith a half tube of lead, except 
for the head which was held in position by two metal clips. A small brass 
cylinder, 1 cm in diameter, was used for local application of x-rays and 




Fig. 5. The same case as Fig. li. Fibrinoid ( plasmatic] ciisorgamzation of greatly 
enlarged angioectatic vessels with erythrodiapedesis and transudation of fibrin- 
containing fluid into the tissue of the posterior horn producing a necrotizing effect. 
Masson stain. 




Fig. 6. (see text) 
216 



MORPHOLOGICAL EFFECT OF X-RAYS TO THE CNS 



217 



positioned on the mid-dorsal skull. Technical conditions included: 40 kv, 
25 ma, filter 0.3 mm Al, and focus-skin distance 5.5 cm. To determine more 
exactly the actual intracerebral radiation, the x-ray dosage was measured by 
a Siemens dosimeter at a level to include skin, bone, and 1 mm of brain 
substance, which means that within a distance of 2.5 mm about 50% of 
the surface dose was measured. Further \alues were obtained by using 1 mm 
plates of a phantom material, Cellon, which has the same absorption value 
as brain tissue. This procedure indicated a diminution of the dose in dif- 
ferent regions (Fig. 7). EflFective x-radiation values of 1,000 to 80,000 r 
were administered to the cerebral cortex with application times of 28 sec to 
37 min 34 sec, respectively. Doses of 20,000 r and more were badly tolerated 
by the animals; generally they died spontaneously 2 or 3 days later. Young 
animals seemed to be more sensiti\e than older ones, and on the average 
showed more sexere morphologic changes. With the application of 30,000 r, 
sharply limited zones of total necroses, sometimes containing numerous small 
hemorrhages, could be produced and were fully de\eIoped after 67 to 68 











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mm 



10 



10 20 30 40 50 
(mm) 



Fig. 7. Equi\alent doses for special tube used with soft x-ray radiation. The diagram 
demonstrates the diminution of the x-ray dose at different levels in the tissue. The 
figures represent the effecti\e dose in percentages of the surface dose. The abscissa 
demonstrates the diffusion of the x-ray beam within the tissue. 



218 



W. SCHOLZ, W. SCHLOTE AND W. HIRSCHBERGER 




Fig. 8. Sharply limited total radionccrosis of semicircular shape in the brain of a 
Syrian hamster, following the application of 30,000 r after 67 hours of survival. 
Numerous diapedetic hemorrhages, some of them far from the necrotic zone in the 
thalamus and midbrain. H. and E. 



hours (Fig. 8). It was not possible to identify the different types of cells in 
the necrotic zone, which included the dorsal part of the thalamus. The 
perikaryon had disappeared and all nuclei, including numerous polymorpho- 
nuclear leucocytes, were in a state of pyknosis or rhexis (Fig. 9). A fairly 
large number of small hemorrhages could be observed at some distance 
from the necrotic zone. No progressive interstitial reaction of the glial or 
mesenchymal tissue was seen. 

It does not seem possible to determine the pathogenesis of these necroses, 
which have been designated as anemic by Russel ct al. (1949) because of 
their pale appearance. We did not find occlusions of pial vessels or larger 
arteries, and certainly the necrosis does not involve a particular region of 
arterial irrigation. Rather, it is restricted to just the irradiated field with a 
semicircular penetration into the depths of the cerebral tissue. To exclude 
an ischemic condition as the cause of the necrosis, India ink was injected 
into the left ventricle of the heart of living anesthetized animals. The freely 
circulating blood carried the indicator substance throughout the capillary 
bed of the area that had received 20,000 r 50 hours before (Fig. 10). In 
some places, where erythrodiapedesis had occurred, the India ink pene- 




y » 



^^V"'- 



• *.., fej.% ^.» ...^ -^^ **- .*/, V •'r. 










. M' 






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Fig. 9. Multiple hemorrhages, pyknosis. and rhexis ol tissue cells and of emigrated 
polymorphonuclear leucocytes in a necrotic region of the same type as in Fig. 8 and 
produced by the same x-ray dose. H. and E. 













Fig. 10. Nearly complete representation of the capillary bed of the cerebal cortex 
by India ink, 50 hours after local irradiation of a Syrian hamster with 20.000 r. 

219 



220 



W. SCHOLZ, W. SCHLOTE AND W. HIRSCHBERGER 



trated into the tissue. This region was completely stained blue with trypan 
blue injected subcutaneously shortly after the irradiation. 

To clarify these observations, the experimental technique was modified 
in two diflferent ways: (1) we diminished the x-ray dose to 1,000 r, and 
(2) we shortened the survival time to 1 hour, using x-ray doses large enough 
to produce complete tissue necroses. With the first of these methods, the 
size of the necrotic area decreased, so that with 5,000 r only a small tan- 
gential zone of the cortex including the first and second layer was affected. 
These lesions developed within about 8 days. The nerve cells and glia cells 
lost their cytoplasm, and their nuclei were shrunken. A few polymorpho- 
nuclear leucocytes were scattered throughout the necrotic tissue. This small 
necrotic zone was surrounded by a broad region of spongy tissue containing 
numerous tiny hemorrhages. Within 6 days, an application of 10,000 r 
produced a larger zone of necrosis extending through the whole cortex 
(Fig. 11). In these cases the first reactive processes had begun, and several 
fat granular cells and progressive glial cells were found at the borders of 
the necrotic tissue, especially near the pia. However, within the center of 
the necrosis nothing seemed viable. Here again, the zone of hemorrhages 
was considerably more extensive than the necrotic area. The necrotic zone, 











i^ 



Fig. 11. Large, sharply bordered areas of acute radionecrosis invohing the whole 
cortex and corpus callosum and containing numerous, partly confluent, diapedetic 
hemorrhages; 10,000 r with 6 days survival. Azan stain. 



MORPHOLOGICAL EFFECT OF X-RAYS TO THE CNS 221 




Fig. 12. Intravital trypan blue staining of the necrotic region of the same brain as 
in Fig. 11. 



even when small, was surrounded by a broad shell of sponoy tissue where 
the cellular elements showed only minor chanoes. To demonstrate the bar- 
rier function, a series of animals recei\ed a subcutaneous injection of 1 cc 
of Kr trypan blue solution from 1 to 6 hours after irradiation. As is well 
known, the brain remains unstained under normal conditions. With disinte- 
gration of tissue and destruction of vessels, the dye may enter the tissue. 
Figure 1 1 demonstrates such a necrotic zone which had developed within 
6 days after x-irradiation with 10,000 r and which involves the whole 
thickness of the cerebral cortex. From the numerous hemorrhages in the 
necrotic zone, trypan blue spread diffusely all through the destroyed tissue 
(Fig. 12) . These cases do not ser\e to illustrate a disrupted barrier function 
unless the blue staining siupasses the necrosis and extends to the surrounding 
spongy area where only minor cellular changes are seen. This could be 
obsened in some cases, but it is difficult to demonstrate. 

Bv shortening the sur\i\al time of the animals, we attempted to observe 
the earliest stages of tissue necroses. A standard x-ray dose of 20.000 r was 
applied within 10 min 36 sec. This dose proved sufficient to produce clear 
necrosis within 24 horns. Surprisinyly. we could produce lesions ot similar 
size with a considerable diminution of the x-ray dose. After 24 to 26 hours, 
a large semicircular zone of obvious necrosis covering the irradiated field 
could be seen extending from the dorsal surface of the cerebral cortex to 
the corpus callosum. With shorter survival times and in older animals, the 
depth of the necrotic area flattened (Fig. 13). Here again, a sponginess of 




Fig. 13. Earlier stage of radionecrosis in the brain of a Syrian hamster, 24 hours 
after the local application of 20,000 r. The tangentially situated zone of clear necrosis 
has a spongy character and is demarcated by a strip of even more pronounced spongi- 
ness. Azan stain. 







r 


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Fig. 14. The same case as in Fig. 13. In the necrotic zone, the cellular constituents 
can scarcely be diflferentiated, with only a few exceptions. The plasma has become un- 
stainable : the nuclei are pyknotic. Some emieiated polymorphonuclear leucocyte;, are 
also seen. Gallocyanin. 

222 



MORPHOLOGICAL EFFECT OF X-RAYS TO THE CNS 223 

the bordering zone with granular breakdown of the astrocytic processes 
could be observed in Cajal preparations. In the necrotic zone, it was difficult 
to identify the different types of cells Fig. 14). Only a few nerve cells could 
still be recognized by their acidophilic cytoplasm. In all other cells, the 
perikaryon became unstainable. Almost all cells, including glial elements, 
demonstrated pyknotic nuclei. Some polymorphonuclear leucocytes could 
also be recognized. With lurther shortening of sia\i\al time to 7 hours, only 
two small zones of spongy loosening of the tissue were seen, situated almost 
symmetrically in both hemispheres (^Fig. 15 j. They were rather sharply 




\» 



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Fig. 15. Two sharply limited small foci of spongy loosening, symmetrically and 
rather superficially located in the cortex. This is an early stage, 7 hours after local 
application of 20.000 r. H. and E. 

limited by intact nerxous tissue. The cellular elements in the spongy zone 
exhibited only minor changes, such as shrinkage with a clear nuclear struc- 
ture (Fig. 16). Sometimes, however, groups of nerve cells showed signs of 
dissolution, including \acuolization of the cell cytoplasm. After local irradi- 
ation with 45.000 r and a survival time of 6 hours, small areas of sponginess 
of tissue of the same type and size reaching from the surface to the 3rd 
cortical layer could be obser\ed Fig. 17). The nerve cells exhibited only 
minor chances, such as diminished affinity for gallocyanin. and leucocytes 
were absent. With low power, this deviation can be more clearly seen as a 



r 
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Fig. 16. The same preparation as in Fig. 15. Bordering zone of the spongy area, 
with tissvic cells showing only miner changes. H. and E. 



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Fig. 
spheres 
method 




17. Sytimicti K aii\- and raciially ariangetl spdiiniiu-ss v.\ tissue in lioth hemi- 
following local x-irradiation of 45,000 r after 6 hours. W. and E. 
. In this zone, the cells appear less stained. 



224 



MORPHOLOGICAL EFFECT OF X-RAYS TO THE CNS 225 

slight pallor in the cortex (Fio. 18) . Hii^h power demonstrates disappearance 
of Nissl bodies in the large pyramidal cells so that the perikaryon is generally 
pale and difTusely stained, whereas the nuclei of the smaller nerve cells 
contain rather coarse chromatin particles (Fig. 19). It is extremely difficult 
to judge the whole tissue situation from deviations in appearance of the 
cortical nerve cells alone. The significance of such deviations seems to re- 
c}uire a special cell study as performed by Krogh and Bergeder ( 1957) using 
the o^allocvanin stain of Einarson and bv Schiimmelfeder (1957) in his 






.^. «•'■.,• rii.'.t.i...'' i. ■,.■>•- * -.,', ,' , ••• '.•,•- • J, ■<'■■■ ;- ■'' : t -* '. .<- 

>• •'*..;» ";•^ i-v/vV.".".?.' is ':..'.■- •- > -,• '■ „ :• . .. .... ■■.;.-•".'*, ' '/^•'.■' h-.,' x'-'^ • ' 




Fig. 18. EquivaK-nt of the spongy region in Fig. 17, stained with the gallocyanin 
method. In this zone, the cells appear less stained. 



histochemical investigations with fluorescent acridin-orange on the nerve 
cells of the cerebellar cortex. With regard to the different types of nerve 
cell changes after irradiation of 5,000 to 20.000 r, they made similar obser- 
vations on cortical nerve cells, and they considered this to be an expression 
of cell necrosis. Certainly, it requires a high level of experience to decide 
from the appearance of the cell alone that cell shrinking with dark staining 
of the perikaryon and a pyknotic nucleus is not reversible and must inev- 
itably lead to necrosis. In cresyl \iolet and gallocyanin preparations, we 
have seen such cells distributed over wide regions including the cortex of 
control animals which had ne\er been irradiated. Their significance is not 



226 W. SCHOLZ, W. SCHLOTE AND W. HIRSCHBERGER 




Fig. 19. Detail of Fig. 18. The large nerve cells are of normal shape; their peri- 
karyon is lightly stained, and the nucleus is not strikingly altered. The nuclei of the 
smaller nerve cells contain rather coarse chromatin particles. 



known, but the experiments of Scharrer (1933) and of Cammermeyer 
(1960) suggest that they can be produced artificially by simple mechanical 
pressure on the cortex. A high degree of swelling and vacuolization of the 
cytoplasin with loss of ribonucleic acids and the occurrence of coarse 
chromatin particles within a nonpyknotic nucleus seem to constitute evidence 
of necrosis. But again, the nerve cell nuclei of rodents may normally have 
rather coarse chromatin particles. The third variety of nerve cell alteration 
referred to by these authors seems to resemble the so-called ischemic type 
of nerve cell change. Certainly, as soon as a breakdown of the nucleus is 
established, death of the cell must be accepted if postmortal processes can 
be excluded. We have observed the cytoplasm of such cells to be acidophilic, 
staining with eosin and even with azocarmine and acid fuchsin, but we 
have had no opportunity to make coinparisons with the results obtained 
by Schiimmelfeder (1957) with acridine-orange. 

Krogh and Bergeder (1957) did not state whether the pyknosis of the 
granular cells is due to a direct influence of irradiation, or if edema of the 
granular layer may play an intermediary role. Schiimmelfeder ( 1957) favors 
a direct influence of ionizing rays. However, it is not easily imderstood why 
the same cause in the same quantity elicits a shrinking one time and a high 



MORPHOLOGICAL EFFECT OF X-RAYS TO THE CNS 227 

grade swelling of the nerve cells another. In approaching this question, it 
seems necessary to consider the condition of the whole tissue concerned and 
not solely the nerve cells, since they are only a part of the tissue, and a 
necrosis of nerve cells alone does not cause tissue disintegration as seen, 
for example, in anoxic selective neuronal necrosis. Only if the oligoglia 
and astrocytic glia are also destroyed, does the continuity of the tissue 
break down. As we ha\e seen in Cajal preparations, astroglia processes 
show a granular decay rather quickly, long before regressive changes in the 
vessel walls are demonstrable. Schiimmelfeder (1957) also mentions disap- 
pearance of ribonucleic acid in glia cells. This would indicate a much more 
severe lesion of the neivous tissue: however, definite tissue disintegration 
needs a certain time to become manifest with clear structural changes. In 
initial stages, when necrosis is not yet clearly apparent, the most reliable 
histologic phenomenon seems to be the immigration of single leucocytes 
into the tissue and the beginning of erythrodiapedesis. We have tried to 
study the earliest stages of cortical necrosis in regard to the function of the 
hematoencephalic barrier. We did not succeed in staining intravitally with 
trypan blue during initial stages of spongy tissue transformation (Figs. 
15-19). when seemingly only minor alterations such as moderate swelling 
and dissolution of the Nissl substance without significant changes of the 
nuclei were seen. A positive result was attainable, however, before distinct 
symptoms of tissue disintegration became e\ident. Thus, the blue staining 
with trypan blue was complete as soon as single diapedetic hemorrhages of 
small size in the zone of irradiation could be observed or single polymorpho- 
nuclear leucocytes had immigrated into the nervous tissue. This is demon- 
strated by a section stained with the azocarmin Malloiy method of Heiden- 
hain (Fig. 20) and, for comparison, by a macro photo of the blue stained 
field on the siufacc of the brain i Fig. 21). 

Discussion 

We must emphasize that the delayed radionecroses of the spinal cord are 
not significantly diflferent from those in the brain. We obserxe the same 
phenomena of altered vascular permeability which in the white matter 
results chiefly in transudation and in the gray matter chiefly in plasmatic 
infiltration, often with erythrodiapedesis. It now seems established beyond 
doubt that these processes are primary to all other destruction of nervous 
tissue in delayed radionecrosis. although in many cases structural changes 
of the vessels are not evident. The same pathogenic mechanism in delayed 
radionecrosis is valid in the human brain, as has been demonstrated by 
Fischer and Holfelder (1933), Markiewicz (1935), Scholz and Hsu (1938), 
Zeman (1949, 1955) and others. In a necrotic zone, e\'en after many years 



228 



W. SCHOLZ, W. SCHLOTE AND W. HIRSCHBERGER 




Fig. 20. Early and incomplete radionecrosis, 20 hours after local x-irradiation with 
20,000 r. Only small single hemorrhages and slight diminution of stainability of the 
cortical tissue at both sides of the median fissure are visible. 




Fig. 21. Intravital staining of the irradiated zone of the brain from which Fig. 20 
is taken. 



MORPHOLOGICAL EFFECT OF X-RAYS TO THE CNS 229 

the plasmatic material can be found as an amyloid-like substance, resistant 
to absorption and behavins; like a foreign body. This substance has not been 
demonstrated in large amounts in the central nervous system of experimental 
animals. Whereas most investigators, including in recent years Berg and 
Lindgren ( 1958). and Zeman (1955), acknowledge this pathogenic mecha- 
nism of the delayed radiolesions, it is much more difficult to explain the 
pathogenesis of the acute radionecrosis occurring within hours following the 
single application of extremely large doses of ionizing radiation of high 
intensity. We did not see convincing morphologic changes either with 80,000 
r after 134 hours or with 45,000 r after 1 /a hours; however, necroses were 
fully developed with 30,000 r after 67 hoins. They cover exactly the field 
of irradiation, are apparently not related to regions of arterial irrigation, 
and do not depend on vascular occlusions. As to the restriction of the lesions 
to the field of irradiation and the morphologic appearance of the nerve cell 
changes, our results are largely in accordance with the observations of Krogh 
and Bergeder (1957) and of Schiimmelfeder (1957), though there was some 
difference in the application and measurement of the x-irradiation. It is true 
that fully developed acute radionecroses closely resemble anemic infarctions, 
but we think that such a designation, used by Russel ct al. ( 1949), is not 
justified, since during the development of the necrosis, the free passage 
through the capillary bed has been made exident by India ink. In these cases, 
we find hemorrhages not only in the region of neciosis but also at some 
distance from the necrotic zone, invading the unaltered nervous tissue which 
was exposed to a lower intensity of irradiation and indicating a distiubance 
in the permeability of the blood vessels. Usually, the centers of the necroses 
are surrounded by a broad shell of spongy tissue which raises the question 
of whether we deal simply with a demarcation zone as seen around every 
more or less complete necrosis. However, the initial stages of such necroses 
produced by an irradiation of 20.000 and 45,000 r after 7 and 6 hours, 
respectively, consist only of such spongy loosened tissue, suggesting a local 
edema and demonstrating no distinct signs of cellular necrosis. Efforts to 
stain these earliest lesions intravitally with trypan blue were not successful, 
probably because the disorder of the blood-brain barrier is still incomplete in 
this stage. But already in the next stage, when only minor changes of the 
cellular constituents of the tissue manifest themselves and only a few vessels 
show beginning diapedesis, we find a distinct blue coloration, pointing to 
the important role of permeability disorders in the pathogenesis of the acute 
radionecroses also. As soon as distinct necrosis is present, the trypan blue 
staining spreads far beyond the necrotic center throughout the whole spongy 
zone. It may therefore be reasonable to consider the sponginess of the tissue 
as well as the multiple hemorrhages within and out of the necrotic center 
as being due to a disturbance of penneability of the blood-brain barrier, 



230 W. SCHOLZ, VV. SCHLOTE AND W. HIRSCHBERGER 

primarily caused by irradiation. Krogh and Bergedcr (1957), who produced 
cerebellar lesions by a high x-ray dosage, did not decide whether the break- 
down of the granular layer is secondary to edema or is a primary lesion. In 
experiments with Co''", Vogel et al. (1958) produced a reversible pyknosis 
of the same gramdar cells. Since we know that the granular layer of the 
cerebellar cortex is rather sensitive to edema, we must consider the possibility 
that it is the radiation-induced edema that produces the granular cell 
changes. However, this does not mean that all tissue changes shoidd be con- 
sidered secondary to the edema-like loosening of the tissue. In the cerebral 
cortex, nerve cells remain resistant to a simple edema for a long time. More- 
over, we failed to find in acute radionecroses the dangerous plasmatic infil- 
tration of tissue that can inhibit oxygen difTusion. As all cellular elements 
demonstrate an early and rapid structural breakdown, the assumption may 
be justified that x-irradiation of high dosage and intensity may cause a co- 
ordinated breakdown of the hcmatoencephalic barrier and a primary destruc- 
tion of all other tissue constituents as well. It seems that in delayed lesions, 
the latent period becomes progressively shorter with an increase of x-ray 
dose and intensity, and the hcmatoencephalic barrier, in common with other 
tissue constituents, is at last simultaneously affected by the increased ionizing 
radiation. 

Conclusions 

X-irradiation of the spinal cords of 36 rabbits produced results similar to 
the previously reported delayed x-ray lesions in the brains of dogs. The 
fractionated application of doses up to 1 1 ,000 r within 40 days and single 
doses of 2,000 r were followed by focal zones of disintegration, mainly situ- 
ated in the white matter, but also affecting the gray substance. These changes 
occurred after latent periods of from 4 to 33 weeks. The fibrinoid disor- 
ganization of the vessel walls, erythrodiapedesis, and infiltration of plasmatic 
material into the central nervous tissue seem to be the primary lesions and 
demonstrate a breakdown of the hcmatoencephalic barrier. All other changes 
of the tissue, including demyelination and breakdown of cellular constituents, 
were preceded by permeability distiubance and may be considered secondary. 
In the brains of approximately 100 Syrian hamsters, acute radionecroses 
were produced by single applications of large x-ray doses of high intensity. 
In animals receiving 5,000 r, necroses were fully developed within 8 days; 
within 6 days after 10,000 r, and within 3 days after 45,000 r. Several animals 
receiving 20,000 r showed distinct disintegration of tissue in the field of 
irradiation after 24 hours, and initial stages in these cases could be detected 
after only 7 hours. The sharply limited semicircularly shaped lesions covered 
exactly the field of irradiation and decreased in depth with the diminution 



MORPHOLOGICAL EFFECT OF X-RAYS TO THE CNS 231 

of the x-ray dose and abbre\iation of the sur\i\al time. In fully developed 
necroses, all cellular elements were broken down, and erythrodiapedesis 
from more or less numerous \essels occurred not only within, but also out 
of the necrotic zone. Initial stages demonstrated a spong\' transformation of 
the nervous tissue with only minor changes of the cellular constituents, such 
as plasma and nuclear shrinkage. Here, erythrodiapedesis was still lacking. 
A disorder of the hematoencephalic barrier in this initially spongy territory 
could not be demonstrated by intra\ital trypan blue staining, while a blue 
coloration of a large area including the surrounding region of spongy loosen- 
ing became visible before a distinct disintegration of tissue, indicated by signs 
of cell necrosis, were demonstrable. Solitary small diapedetic hemorrhages 
and the immigration of single leucocytes seem to indicate a degree of perme- 
ability disorder sufficient to allow an intraxital staining with trypan blue. 
This beha\ior and the appearance of more or less small hemorrhages at the 
borders of. and sometimes far from, the necrotic foci point to the significance 
of an early disorder of the blood-brain barrier. Since ner\e cells have been 
shown to be rather resistant within zones of edematous loosening of tissue, 
and astrocytes even may become progressi\e, the whole process of necrosis 
cannot be considered as solely secondary to the permeability disorder. From 
the morphologic tacts, it seems justified to admit that in acute radionecrosis 
from large x-ray doses ot hiizh intensity a direct effect on the nervous and 
astrocytic tissue constituents may occur simultaneously with \ascular damage. 
Processes of transformation, resorption, and organization de\elop slowlv and 
are established regularly onlv at the borders of the necrotic zones. 

References 

Berg. N. O., and Lindgren. M. 1958. Time-dose relationship and morphologv- of 

delayed radiation lesions of the brain in rabbits. Acta Radiol. Suppl. 167. 
Cammermeyer, J. 1960. A critique of neuronal hyperchromatosis. /. Xeuropathol. 

Exptl. Neurol. 19, 141-142. 
Fischer, A.. W., and Holfelder. H. 1933. Lokales .Amyloid im Gehirn. Deut. Z. Chir. 

227, 475. 
Krogh, E. v., and Bergeder. H. D. 1957. Experimental irradiation damage of the 

cerebellum demonstrated by Einarson's Gallocyanin-chromalum staining method. 

/"" Congr. intern. Sci. Neurol., Brussels, 1957: 3' Congr. intern. Neuropathol. 

pp. 287-294. -Acta Medica Belgica, Brussels. 
Lyman, R. S., Kupalov, P. S., and Scholz, VV. 1933. Effect of Roentgen rays on the 

central nervous system. A.M. A. Arch. Neurol. Psychiat. 29, 56-87. 
Markiewicz, T. 1935. Uber Spatschiidigungen des menschlichen Gehirns durch Ront- 

genstrahlen. Z. ges. Neurol. Psychiat. 152, 548-568. 
Mogilnitzky. B. N., and Podljaschuk, L. D. 1930. Rontgenstrahlen und sogen. '"hama- 

toenzephalische Barricre." Fortschr. Gehiete Rontgenstrahlen 41, 66. 
Russell. D. S.. Wilson. C. \\'., and Tansley, K. 1949. Experimental radionecrosis of 

the brain in rabbits. /. Neurol. Neurosurg. Psychiat. 12. 187. 



232 W. SCHOLZ, W. SCHLOTE AND W. HIRSCHBERGER 

Scharrer, E. 1933. Bemerkungen zur Frage der "sklcrotischen" Zellen im Tiergehirn, 

Z. ges. Neurol. Psychiat. 148, 773-777. 
Scholz, W. 1935. Uber die Empfindlichkeit des Gehirns fiir Rontgen- und Radium- 

strahlen. Klin. Wochschr. 14, 189-193. 
Scholz, W., and Hsii, Y. K. 1938. Late damage from Roentgen irradiation of the 

human brain. A.M. A. Arch. Neurol. Psychiat. 40, 928-936. 
Scholz, W., Ducho, E.-G., and Breit, A. 1959. Experimentelle Rontgenspjitschaden am 

Riickenmark des erwachsenen Kaninchens. Psychiat. et Neurol. Japan. 61, 417-442. 
Schiimmelfeder, N. 1957. Fluoreszenzmikroskopische und cytochemische Untersuchun- 

gen iiber Friihschaden am Kleinhirn der Maus nach Rontgcnbestrahlung. 1" Congr. 

intern. Sci Neurol., Brussels, 1957: 3' Congr. intern. Neuropathol. pp. 295-308. 

Acta Medica Bclgica, Brussels. 
Vogel, F. S., Hoak, C. G., Sloper, J. C., and Haymaker, W. 1958. The induction of 

acute morphological changes in the central nervous system and pituitary body of 

macaque monkeys by cobalt'" (gamma) radiation. /. Neuropathol. E.xptl. Neurol. 

17, 138-150. 
Zeman, W. 1955. Elektrische Schadigungen und Vcranderungen durch ionisierende 

Strahlcn. In "Handbuch der speziellen pathologischen Anatomie und Histologic" 

(O. Lubarsch et al, eds.). Vol. XIII, Part 3, pp. 327-362. Springer. Berlin. 
Zeman, W. 1949. Zur Frage der Rontgenstrahlenwirkurg am tumorkranken Gehim. 

Arch. Psychiat. Nervenkrankh. 182, 713-730. 



A Demyelinating or Malacic Myelopathy 

and Myodegeneration— 

Delayed Effect of Localized X-irradiation 

In Experimental Rats and Monkeys 

J. R. M. Innes and a. Carsten 

Brookhaven National Laboratory 
Upton, Long Island, New York 



Introduction 

For many years the central nervous system was considered hislily resistant 
to radiation damaiie — and statements to this effect occasionally still appear. 
It is manifest that there must be some ciualification by reference to the part 
of the system exposed, the conditions and dosimetry of irradiation, and the 
species and age of the animals used. The earlier experimental irradiation 
work on the normal nervous system was reviewed by Warren (1943) and 
Hicks f 1952 1. We are concerned here with oiu initial experimental irradi- 
ation studies on the spinal cord of rats and with some observations on 
experimental monkeys. 

In man the hazard attached to x-irradiation ot the brain or spinal cord, 
whether by deliberate design for radiotherapy or unaxoidably when extra- 
neural sites must be exposed, is well established. Late or delayed irradiation 
effects on the nerxous system are different from acute massixe radionecrosis 
which follows extremely high doses. The problems associated with the two 
types of damage are multiple and complex, and the literature was reviewed 
by Zeman ( 1955) and by Zollinger 1960) . Many original papers on human 
cases have been perused, e.g., Lyman ct al. (1933), Stevenson and Eckhart 
(1945), Pennybacker and Russell (1948). Greenfield and Stark (1948), 
Boden (1948), Friedman i 1954 1, Itabashi et al. (1957), and Dynes and 
Smedal ( I960). It is not necessary to deal with these contributions individ- 
uallv. but we can reiterate the patholologic problems to be faced. The 
number of reported cases in the literature is an inde.x neither to the inci- 
dence of delayed irradiation lesions in the spinal cord, nor to the importance 
of the hazard, as is evident from discussions with neurologists, neuropathol- 

233 



234 J. R. M. INNES AND A. CARSTEN 

ogists and radiotherapeutists. As Itabashi ct al. (1957) pointed out, when a 
neoplasm has been the target for irradiation, neurologic signs which might 
appear later have usually been attributed either to metastasis or extension 
of the primary lesion, especially when there has been some apparent im- 
provement. Further, many human cases may not be followed up after 
x-irradiation of the spine; in others, autopsies may not be possible when 
death occurs years later, or it may not be possible to examine the spinal cord 
at autopsy. Some papers on delayed irradiation myelopathy are based largely 
on clinical data, and the lesions have not been comprehensively studied. 
Dynes and Smedal's series included 10 therapy cases, and Friedman (1954) 
estimated that the incidence of delayed neurologic damage was 10% in 
100 patients with testicular carcinoma whose spinal cord had received 
5,000 rads or more. In all cases, subsequent to irradiation, there is a latent 
period ranging from many months to many years, during which there are 
no neurologic signs or symptoms due to irradiation. The clinical onset can 
be abrupt or insidious, with a variable neurologic syndrome leading to 
paraplegia and inevitably to death, although some patients have lived for 
years with paraplegia (Dynes and Smedal, 1960). 

Lesions in the spinal cord in such cases have been variously reported as 
radionecrosis, postirradiation myelitis, or myelopathy; but whatever the 
designation, the damage can be devastating and appears no different from 
that described by us (cf. Itabashi et al., 1957, one of the few papers describ- 
ing spinal cord damage in man). In the delayed postirradiation process, it is 
not the most superficial layers of nervous tissue which are most radiosensitive, 
but the white matter in both brain and spinal cord. Neuroglial response has 
varied according to different reports, but it can be absent or negligible, and 
in some late lesions neuroglia must have been destroyed at a rate equal to 
the damage to white matter or rendered incapable of response. In both the 
human brain and cord, observers have commented on the difficulty of 
separating the damage done specifically by the irradiation on tissue already 
traumatized by another cause, such as a tumor. 

The fundamental question, still not answered, is whether the damage is 
a direct effect of the radiation or an indirect one caused by a primary 
change in vascular walls which leads to interference of the normal blood 
supply, possibly then emanating from chronic hypoxia or ischemia. If it 
were a direct effect, then the question arises as to what happens to the 
neural tissue in the latent period before the eventual neurologic signs and 
symptoms. Years ago, Scholz (Scholz et al., 1959) and Zeman (1955) ob- 
served in such late lesions the deposition in and around vessels of an 
"amyloid or paramyloid material," the nature of which has never been 
unequivocally established by histochemical methods. The thickening of 
vessel walls and subsequent constriction of lumina were thought to cause 



X-IRRADIATION AND DELAYED MYELOMALACIA 235 

hypoxia or dynamic alterations in the \ascular walls resultins: in increased 
capillary permeability and seeping through of plasma. This was considered 
sufficient to account for the lesions in areas of neural substance supplied by 
the afi'ected vessels. These themes have been discussed repeatedly (see 
Zeman, 1955, and the most recent paper by Scholz et al., 1959). 

It is doubtful if experimental work has helped greatly, whether we are 
dealing with brain or spinal cord ( Malamud vt al., 1954; ( Pennybacker and 
Russell, 1948: Warren. 1943; Davidoff et al, 1938; Clemente and Hoist, 
1954; McLaurin rt «/., 1955; Scholz ct al., 1959). Nor are the reasons hard 
to find. The acute necrosis produced by massi\e doses of x-rays does not help 
to explain the pathogenesis of the late delayed damage. After x-irradiation, 
experimentalists ha\e noted the utterly unpredictable variations (a) of the 
reaction in difl'erent animals of the same species and age, some animals 
remaining unscathed under the same experimental conditions and dosimetry 
which causes marked late lesions in others, lb) in the latent period before 
nemologic signs de\elop. and c ) in the occurrence of the \ascular changes, 
because thickening or deposition of "amyloid material" has not always 
been observed to be associated with the neural lesion. 

An experimental approach with any animal species brings out that it 
is different than working with established transmissible neurotropic infec- 
tions which can be reasonably controlled — the dose of causal agent related 
to a regular incubation period and a specific pathologic effect. The \ arying 
latent periods (Table I) set a formidable barrier in designing an experiment 
on a quantitative basis. Many animals may not develop neurologic signs or 
lesions under the same conditions of experiment, and to determine this with 
certainty, it might be necessary to wait much longer than 1 year. Little 
progress toward the solution of the problem may be expected until a lesion 
can be produced consistently in small animals, under controlled conditions 
related to dosimetry and exact localization of exposure. Monkeys and dogs 
can hardly be used in large numbers because of the expense invoked, 
although delayed cerebral and spinal cord lesions have been produced in 
both species, and more meticulous clinical obser\ations are f)ossible with the 
larger animals. If this were possible, it might open the way for study of the 
pathogenesis of the myelopathv by examination of a large series of animals 
from a few days postirradiation up to a year or more. Of ecjual importance 
would be more accurate determination of the minimal pathologic dose to 
effect the spinal cord damage. Our obser\ations were made with these facts 
in mind, and their importance may lie in the consistency of production of 
spinal lesions in rats, together with the continuing study of the pathogenesis. 

The work was initiated by obser\ations on rats exposed to upper body 
irradiation in which some clinical signs suggested the animals might be 
suffering from myelitic pain, evidenced by irritability, sensiti\ity to touch, 



236 J. R. M. INNES AND A. CARSTEN 

and changes in beha\ior. Some animals developed paralysis of the hind legs 
many months after irradiation. After identification of the demyelinating 
myelopathy in a few rats, a series of experiments were planned in which, 
by adequate shielding, only the vertebral column from about sixth cervical 
to second thoracic segment was irradiated. The first experiments were con- 
cerned with definition of the pathologic process and its topographic neuro- 
anatomic distribution. 

Techniques 

White female rats were housed two in a cage and given food and water 
ad libitum throughout the experiment. They were 3 to 6 months of age at 
irradiation. Before and after irradiation, they were weighed and examined 
daily. 

Rats were anesthetized with intraperitoneal injections of sodium pento- 
barbital, 45 mg per kg of body weight. In the groups exposed to upper 
body irradiation, the animals were marked with dye at the xiphoid process 
and placed in 2-in. diameter lucite tubes. A ^4 -in. -thick cylindrical lead 
shield was placed around the lower half of the tube at the dye mark, so 
that the upper body and head of the animal were exposed to the x-ray beam. 
In rats receixing thoracic exposure, a second lead shield was placed over 
the head end of tlie lucite tube at the manubrium. Animals indicated as 
spine exposed were completely surrounded by ^ in. lead shields containing 
a small hole in a position so that only the spine from sixth cervical to 
second thoracic segment was exposed (Table I). 

Tlie irradiations were made with a General Electric 250 kV Maxitron 
x-ray machine. The radiation factors were: 250 k\p. 30 ma. 0.5 mm Cu 
filter. Aluminum parabolic filter for field uniformity, target to skin distance 
from 6.5 to 9 in., dose rate of 200 to 350 rad per minute measured in a 
tissue ecjuixalent phantom, half \alue layer of 2.15 mm of copper. All 
animals rats and monkeys) received 3500 rads. 

Clinical Findings 

After irradiation, a latent period varying from 5 to 9/2 months occurred 
in rats. The first sign of untoward involvement of the spinal cord was in- 
continence of urine, which persisted to some degree in some rats for weeks 
before any motor weakness or incoordination of the hind legs developed. 
Thereafter, the rats became obviously unsteady on their hind feet and were 
ataxic, and the tail lost its tonicity. This syndrome progressed imtil the 
animal's hind quarters were completely immobilized, without the tail or 
limbs becoming completely flaccid. An afTected animal could move around, 



X-IRRADIATION AND DELAYED MYELOMALACIA 

TABLE I 
Experimental Rats 



237 









Duration of clinical 




Irradiation 


Latent period 


signs 


until sacrifice 


Rat number 


(3500 rads) 


(months) 




(days) 


1 (42/59) 


thorax 


5 




3 


2 (70/59) 


thorax 


^y. 




24 


3 (96/59) 


spine 


VA 




2 


4 (9/60) 


spine 


5 




4 


5 (11/60) 


spine 


5 




28 


6 (17/60) 


thorax 


7 




2 


7 (18/60) 


thorax 


5'/. 




2 


8 (21/60) 


spine 


7 


No 
but 


clinical signs, 
lesions in 








spinal cord 


9 (27/60) 


spine 


6'/. 




8 


10 (67/60) 


thorax 


9 




28 


11 (81/60) / 


spine 


9'/. 


No 


clinical signs 



pulling itself by its forelimbs with the hind limbs cliat:L;inu helplessly behind. 
Complete loss of sphincter control of bladder and rectum resulted in the 
hind charters becoming permanently wet and soiled. The "paralyzed"" rats 
were allowed to surxive for a few days up to 28 days beiore being sacrificed 
for neuropatholosic study lable I ' . 

Pathologic Findings 

As a routine survey for lesions and their extent and distribution, the spinal 
cord was fixed in situ in the \ertebral cokmin with its sin rounding skeletal 
muscles. Subsequent to fixation and decalcification, the column was sliced 
trans\ersely throughout its entire length in 1-2 mm pieces. The caudal 
surface of each slice was sectioned and stained with hematoxylin and eosin. 
Usually this amounted to 28 or more blocks i including usually 3 of the 
brain i being cut from each rat. The distribution of the lesions was plotted 
for each rat as in Fig. 5. We do not include any detailed reference to 
examination of the spinal cord of the many rats irradiated and killed at 
inter\als from 1 dav to 1 month, or to extensive histologic and histochemical 
work on embedded and frozen sections. The spinal cords from nonirradiated 
control rats were also studied. 

As might be expected from the variability of the latent period and the 
time allowed between on.set of signs and sacrifice, there was some varia- 
bility in extent of damage to the spinal cord. 



238 



J. R. M. INNES AND A. CARSTEN 










r^' 




X-IRRADIATIOX AND DELAYED MYELOMALACIA 239 

At the level in the cord where the lesion was seen in its fullest develop- 
ment (Figs. 1, 2. and 3), the process had de\eloped into acute malacia 
terminating in severe liquefaction of the white matter. To some extent, the 
ventrolateral white columns were more damaged than the dorsal columns, 
for although dorsal and \entral areas were affected in some rats ' e.g. Fig. 1 ) , 
the dorsal columns were never selectively changed. In paraffin sections, the 
afTected areas were spongy or reduced to holes and cystic spaces, bridging 
across which were scattered skeins of glial and reticular fibrils and minute 
vessels. As Figs. 1-3 show, the process constantly appeared more severe 
under the leptomeninx, gradually decreasing in intensity inwards. In most 
rats the gray matter was intact and never showed acute softening as in the 
white substance. In the spongy and cystic areas, there were no gitter cells. 
or so few as not to be noticeable, nor was there any astrocytic or fibrillary 
response — glial or reticulai' > Fig. 4 ) . Parts of fragmented axis cylinders 
were scattered throughout the malacic focus, sometimes within what was 
presumed to be ballooned and liquefied myelin sheaths. There was no 
meningeal reaction, no hemorrhage, and the spinal nerves and ganglia in 
the same area were undamaged. 

The lesion described corresponded in its regional distribution to the 
irradiated area of the body. For e.xample. in animals in which the thorax 
had been irradiated, only the thoracic cord was afliected. In others, where 
the spine was selectively irradiated from about sixth cer\ical to second 
thoracic segment, only that area showed the acute damage. Above and below 
the focus, secondary iWallerian) degeneration was clearly e\ident. depicted 
in the pariffin sections by numerous "holes" — i.e., liquefied myelin sheaths 
and axis cylinders. 

As an example of distribution of the lesions in a thorax-irradiated rat. 
Fig. 5 is a diagram of slices of the spinal cord cut at different le\els. As 
sections are studied starting at the first cervical and proceeding in series to 
the sacral level, normal spinal cord and \ertebral marrow is lound until 
the irradiated area is reached. The lesion then may start on one side, then 
the other, and continues until it merges into an irreyular funicular focus of 



Fig. 1. Spinal cord, rat. spine irradiated. Latent period 6' _• months, sacrificed after 
8 days duration of neuroparalysis. Severe myelomalacia of all parts of white matter — 
dorsal and ventrolateral columns. Fatty marrow. Lesions in muscle (upper left) not 
too clear at this magnification (See Figs. 7.A and 7B). Hemato.xylin-eosin. .\bout 
X 16. (.Area marked by arrow shown in Figure 4). 

Fig. 2. -Another case, rat (17/60). thorax irradiated. Latent period 7 months, 
duration of signs 2 days. Set' Fig. 5 for distribution of lesions. Malacia confined at 
this level to lateral column of white matter on one side. Fatty marrow. Hematoxylin- 
eosin. X 16. 



240 



J. R. M. INNES AND A. CARSTEN 










K'. .•■; 









n /' 



■■ 4e i 



fc,:** '.■'■ '■> 



. z;^ '/f? 



?/V 



'C'^ 






(5- c,.* 






'^'m&^^M.'- 



X-IRRADIATION AND DELAYED MYELOMALACIA 241 

malacia. This is related to the well known irradiation damage which affects 
the marrow of the surrounding vertebrae. 

Two rats that showed no clinical signs were killed at 7 and 9/2 months 
after irradiation, and small lesions (Fig. 6) were found in the white matter 
at an early stage of development. 

Severe myodegeneration and necrosis of the veitebral skeletal muscles in 
the same area as the spinal cord damage was almost a constant concomitant 
finding (Figs. 7 A and 7B). Such changes have not been reported in experi- 
mental studies by others or in human cases of postirradiation myelopathy. 

Experimental work on x-irradiated monkeys 

Concurrently with the rat studies, comparable experiments were carried 
out on adult monkeys, and they will be reported upon separately (neuro- 
pathologic studies made in collaboration with Webb Haymaker, M.D., 
Armed Forces Institute of Pathology, Washington, D.C.). These can be 
briefly summarized. Five monkeys were irradiated by the same method and 
exposures (3500 rads) were restricted to the same area of the vertebral 
column as in the rats, i.e. to include the area from the sixth cervical to 
second thoracic segment of the spinal cord. 

One monkey died from pneiniionia 3 months 3 days after irradiation 
without showing neurologic signs, and no lesions were found in the irradi- 
ated part of the spinal cord. One monkey developed neurologic signs 5 
months 13 days after the irradiation; motor weakness of the lower limbs 
started and progressed until there was complete paralysis without the legs 
or tail being flaccid. There was also loss of sphincter control. In spite of 
this, the monkey remained very agile and climbed around its cage and tree 
by use of the arms alone. The animal was sacrificed for study of the nervous 
system after a clinical course of 4 weeks. A malacic myelopathy similar to 
that in experimental rats was found in the irradiated part of the spinal cord. 
Two of the monkeys developed neurologic signs between 6 and 61/2 months 
postirradiation. Another monkey developed signs 8 months 13 days after 
irradiation, but the paralytic course was thereafter very acute and the animal 
was killed when moribund, 4 days after the onset of clinical signs. 



Fig. 3. Another rat. Latent period 5yi months, duration of cHnical signs 2 days 
before sacrifice. At this level, the lesion is more pronounced on one side of ventro- 
lateral column than the other. Hematoxylin-eosin X 16. 

Fig. 4. High magnification from area in Fig. 1 marked by arrow. Pia mater and 
nerve roots on right; gray matter, left. Almost complete tissue dissolution of white 
matter witli no neuroglial response. Hematoxylin-eosin. X 100. 















13 




14 





Fig. 5. Same case as Fig. 2, showing distribution of lesions in the cord. Malacia 
represented by white areas. Secondary degeneration above and below shown by 
smaller scattered white blobs, from No. 5 to about No. 9. Lesion starts about No. 9 
and develops to its maximum intensity about No. 12, fading out about section No. 14. 
Numbers 1-8 roughly represent the cervical cord and its segmentation; the remainder, 
thoracic cord. 



242 



X-IRRADIATION AND DELAYED MYELOMALACIA 



243 




Fig. 6. Spinal cord of rat spine irradiated. Killed alter 7 months with no neuro- 
logic signs, .\bout C 8-T 1. Early stage of small malacic focus about C 8-T 1 on 
the left. Fatty marrow. Hematoxylin-eosin. X 16. 

Discussion 



A severe myelopathy, localized to the area of irradiation, can be produced 
with some consistency in rats, at a dose le\el within the therapeutic range 
used in man. and in which neuroparalytic accidents have occurred after 
deliberate or accidental exposure of the spine. In rats, there is also the par- 
allel of an unpredictable latent period, sometimes many months before the 
onset of progressive neurologic signs. The e.xtent and localization of the 
myelomalacia in the rats is thus responsible for the syndrome starting as 
motor weakness of the hind legs and progressing to ataxia and paralysis. 
The clinical pictiue is thus what is commonly called "posterior paralysis" 
in animals, which bv itself means little. Neurologic examination of small 
laboratory animals is restricted to obser\ations on a few cardinal objective 
signs, and may be no indication of what is ultimately found after neuro- 
pathologic studies. For example, in the course of this experimental work and 
by virtue of extensive histologic work, we found pituitary chromophobe 
adenoma in 3 rats, one spinal ependymoma, one sarcoma, and more recently 



244 



J. R. M. INNES AND A. CARSTEN 





















Fic. 7. A. \ ritflual skeletal muscle from same rat as P'ig. li, shdwiiiy nccio^is, waxy 
degeneration, loss of muscle nuclei in center, and some proliferation at periphery of 
lesion. B. Another case with waxy degeneration, calcification and early fibrosis. 
Hematoxylin-eosin, X 80. 

a spinal oli,s;odendros;lionia (Innes and Borner, 1961). Apart from some 
head tilting and circling in the rats with the cranial tumors, all showed 
virtually a comparable clinical picture. 

The malacic lesion occurring after a long latent period and due to 



X-IRRADIATION AND DELAYED MYELOMALACIA 245 

x-irradiation of the spinal cord in experimental rats is different from the 
ravaging necrobiosis wreaked on all areas of the spinal cord (gray and white 
matter) when the animals are exposed to extremely high doses, with survival 
from a few days to a week. From the few neuropathologic studies on human 
cases (Itabashi ct ai, 1957), delayed irradiation myelopathy in rats appears 
to be a similar process, with the exception of the absence in rats of unmistak- 
able changes in the wall of vessels. In the spinal cord, white matter seems 
more sensitive than gray. Irradiations could possibly ha\e some direct effect 
on myelin sheaths and axis cylinders, aside from any changes caused in the 
endothelium or walls of vessels. This might be suggested by the work of 
Leboucq (1934). on the inhibitory effect of irradiation on the developing 
myelin of baby rats. 

There might be some concern about the definition of the process, whether 
it should be designated a demyelinating or malacic one. It is demyelinating 
in the sense of predilection for attack on the white matter, and no doubt 
it could start as such. However, it seems that once the lesion starts it is a 
rapidly progressive one and then it cannot be regarded as anything but a 
\ery severe liquefacti\e process. A large variety of histochemical methods 
on sections through such a lesion failed to identify with precision any specific 
degradation products of myelin breakdown. The almost negligible glial reac- 
tion is of importance, and in the white matter the oligodendroglia seem to 
disappear as fast as the myelin sheaths. 

Regarding the changes in the walls of vessels, which ha\e been depicted 
as hyaline, amyloid, or para-amyloid degeneration in human postirradiation 
myelopathy (Zeman. 1955: Scholz ft ai, 1959: Pennybacker and Russell, 
1948) but which were not found by O'Connell and Brunschwig, (1937) 
it is important to note there was no evidence in the rat lesions of the depo- 
sition of any imusual degenerati\e substance in or around vessels. Whether 
dynamic alterations in capillary permeability are responsible for an irre\o- 
cable destruction of white matter is another problem. 

A spontaneous demyelinating disease of the spinal cord ! and not the 
brain) in two rats was reported by Pappenheimer i 1952). From his descrip- 
tions and illustrations, the condition cannot be distinguished clinically or 
pathologically from experimental postirradiation myelopathy. We know that 
comparable types of disease processes can be produced in the ner\ous system 
by divergent types of causal agents; but that spontaneous demyelinating 
myelopathy can also occur in rats, should be recognized. Pappenheimer was 
unable to transmit the disease to other rats or mice: the cause was never 
ascertained, nor has any similar disorder been reported by others. Despite 
this, we cannot relegate into the limbo of forgotten things the remote feasa- 
bility that Pappenheimer's murine disease was caused by a virus and that 
irradiation might not light up some latent neurotropic infection. 

There remains to be considered the degeneration and necrosis of the 



246 J. R. M. INNES AND A. CARSTEN 

vertebral muscles in the direct vicinity of the spinal cord damage, i.e., in 
the same irradiated area. The specificity of this myodegeneration in that it 
was caused by the irradiation is undoubted for the lesion is certainly not 
artifact or traumatic due to handling the rats. The changes are no different 
in kind or severity from those seen in types of myodegeneration of man, 
domestic, or laboratory animals, and which can be produced by a multi- 
plicity of causes, perhaps most characteristically in natural and experimental 
alpha-tocopherol deficiency (Hadlow, 1961 ). That some of the lesions were 
old chronic ones was e\ident by the frecjuency of calcareous depositions, and 
again no changes in the walls of arteries supplying aflfected muscles were 
seen. Skeletal muscle is regarded as radio-resistant, but there are few obser- 
\ations on muscle in concurrent studies of any more deep-seated process 
which follows irradiation of the nervous system. 

The study is being continued using both rats and monkeys, with the 
]3articular aim of seeking clues to determination of the early stage of 
damage to the nervous system and thus to a better understanding of patho- 
genesis. The clinico-pathologic studies on monkeys along with serial EEG 
recordings is but part of this study. In rats, groups of animals have also 
been irradiated in the lumbar enlargements of the spinal cord. Finally, as 
such experimental work has clear medical radiotherapeutic implications, 
groups of animals are now being irradiated with the same dose (3500 rads), 
but in divided doses following patterns used in radiotherapeutic treatment 
of lumian beings. 

ACKNOWLEDGMRNT.S 

Our thanks are due to Mr. R. F. Smith, Photographic Division, Brook- 
haven National Laboratory, for the photographs; to Miss Claire M. Lallier, 
for technical assistance and care of the experimental rats: and to Miss Ruth 
Wright, for her part in the extensive histological work involved in the 
pathologic studies on the rats. 

Refp:rences 

Boden, G. 1948. Radiation myelitis of the cervical spinal cord. Brit. J. Radiol. 21. 
464-469. 

Clementc, C. D., and Hoist, E. A. 1954. Pathological changes in neurons, neuroglia, 
and blood-brain barrier induced by x-irradiation of heads of monkeys. A.M. A. 
Arch. Neurol. Psychiat. 71, 66-79. 

DavidofT, L. M., Dyke, C. G., Elsberg, C. A., and Tarlov, I. M. 1938. The eflfect of 
radiation applied directly to the brain and spinal cord I. Experimental investiga- 
tions on Macacus rhesus monkeys. Radiology 31, 451-463. 

Dynes, J. B., and Smedal, M. I. 1960. Radiation myelitis, Am. ] . Roentgenol., Radium 
Therapy Nuclear Med. 83, 78-87. 



X-IRRADIATION AND DELAYED MYELOMALACIA 247 

Friedman. M. 1954. Calculated risks of radiation injury of normal tissues in treat- 
ment of cancer of the testis. Proc. 2nd \atl. Cancer Conf. L 390-400. 

Greenfield. M. M.. and Stark. F. \L 1948. Po:t-irradiation neuropathy. Am. J. Roent- 
genol. Radium Therapy 60, 617-622. 

Hadlow, \V. J. 1961. In "Comparative Neuropathology" (J. R. M. Innes and L. Z. 
Saunders, eds.). Chapter 5. Academic Press. New ^'ork. in press. 

Hicks. S. P. 1953. Effects of ionizing radiation on adult and embryonic nervous sys- 
tem. Research Pubis. Assoc. Research Nerrous Mental Disease 32. 439-462. 

Innes. J. R. M., and Borner, G. 1961. Tumors cf the central nervous system of rats: 
with rejjorts of two tumors of the spinal cord and comments on posterior jaaralysis. 
/. Xatl. Cancer Inst. 26. 719-726. 

Innes, J. R. \I., and Saunders, L. Z. 1961. ■■Comparati\c Neuropathology," Chapter 
23. Academic Press. New ^'ork. in press. 

Itabashi, H. H., Bebin, J., and Dejong, R. N. 1957. Postirradiation cer\ical myelo- 
pathy, report of two cases. Neurology 7, 844-852. 

Leboucq. G. 1934. Actions des rayons x sur la formation de la mycline chcz le rat 
blanche. Rev. beige sci. med. 6, 383-387. 

Lyman. R. S., Kupalov. P. S., and Scholz. \V. 1933. Effect of roentgen rays on the 
central ner\ous system. A.M. A. Arch. Seurol. Psychiat. 29. 56-87. 

McLaurin. R. L.. Bailey. O. T.. Harsh. G. R,. HI. and Ingraham. F. D. 1955. The 
effect of gamma and roentgen irradiation on the intact s])inal cord of the monkey. 
Am. ]. Roentgenol., Radium Therapy Nuclear Med. 7.3. 827-835. 

Malamud.N., Boldrey, E. B., Welch. W. K.. and Fadell. E.J. 1954. Necrosis of !irain 
and spinal cord following x-ray therapy. /. Neurosurg. 11, 353-362. 

OConnell, J. E. .\.. and Brunschwig. \. 1937. Obser\ations on the roentgen treat- 
ment of intracranial gliomata with especial reference to the effects of irradiation 
upon the surroimding brain. Brain 60, 230-258. 

Pappenheimer, .-X. M. 1952. Sjjontaneous demyelinating disease of adult rats. Am. J. 
Pathol. 28, 347-355. 

Pennybacker. J., and Russell, D. S. 1948. Necrosis of the brain due to radiation 
therapy. /. Neurol. Neurosurg. Psychiat. INS.] 11. 183-198. 

Scholz, \V.. rjucho, E.-G.. and Breit. .\. 1959. Exprrimentelle Rontgenj]jatschaden 
am Riickenmark des erwachsenen Kaninchens: Ein weiterer Beitrag zur wirkungs- 
weise ionisierende Strahlen auf das Zentralnervose Gewebe. Psychiat et Neurol. 
Japan 61. 417-442 

Stevenson, L. D.. and Eckhart. R. E. 1945. My(4omalacia of the cervical portion of 
the sjjinal cord, [probably the result of roentgen therapy. A.Al.A. Arch Pathol. 39. 
109-112. 

Warren. S. 1943. Effects of radiation on normal tissue \'III. Effects on the gonads 
IX. Effects on the nervous system. A. MA. Arch. Pathol. 35. 121-139. 

Zeman. W. 1955. Electrische Schadigungen und Veriinderungen durch ionisierende 
Strahlen. Erkrankungen des zentralen Ner\ensystems. In "Handbuch der speziellen 
pathologischen .Anatomic und Histologic" (O. Lubarsch et al., eds.). Vol XIII, 
Part 3, [jp. 327-362. Springer. Berlin. 

Zollinger, H. U. 1960. Radiohistologie und Radio- Histopathologic. In "Handbuch 
der .Allgemeinen Pathologic. Strahlung und Wetter" ( F. Biichner et al.. eds.), 
\'ol. X. Part 1. I)]). 127-287. Springer. Berlin. 



Effects of High-Dose Gamma Radiation on 
the Brain and on Individual Neurons* 



F. Stephen Vogel 

Nezr York Hospital — Cornell University Medical Center, 
Xeiv York, Xe;c York 



Massive doses of ionizins, radiation reoularly and promptly brina; about 
characteristic morpholosic alterations in certain neural tissues and in the 
mesenchymal structures in and around the brain (Arnold et al., 1954; Hay- 
maker <'^ al, 1958; Voa;el ct al., 1958: Wilson, 1960). Most notable among 
these chanoes are contraction and pyknosis of the nuclei of the granule 
cells of the cerebellum and leiicocytic infiltration into the walls of the 
cerebral blood vessels, in the leptomeninges, and choroid plexuses. These 
were conspicuous in monkeys exposed to large doses of gamma radiation 
from Ba""-La^^" and Co''" sources, as have been described elsewhere 
(Haymaker ct al.. 1958; Vogel rt al., 1958). 

There is much e\ idence that the pyknotic change in the cerebellar granule 
cells in cats i Briinner. 1920), monkeys Vogel ft al., 1958), rabbits 
(Gerstner ct al., 1956), guinea pigs ( AKord and Brace, 1957), mice, and 
rats (Hicks and Wright, 1954) is transitory, and ancillary studies indicate 
that similar transient structural changes occur in these cells grown in tissue 
culture and exposed to ionizing radiation. Ne\ertheless, recent observations 
have made it clear that in dogs this cellular response, although initially 
characterized by nuclear contraction, is often followed promptly by karyor- 
rhexis with cellular death (Vogel, 1959 i. When examined with the electron 
microscope, the pyknotic and kaiyorrhetic cells regularly show distinctive 
alterations in intracellular fine structure i Vogel, 1959). These pro\ide in- 

* These studies were conducted at the Uni\ersity of California. .-Xrmed Forces 
Institute of Pathology, Washington, D.C., Randolph .\ir Force Base. Randolph Field, 
Texas, and at the University of Texas and the U.S. .\ir Force, Austin, Texas. They 
were supported by funds provided by the U.S..\.F. School of .Aviation Medicine, Ran- 
dolph .\ir Force Base. Texas, by the Medical Research and Development Board, Office 
of the Surgeon General, U.S. .\rmy. and by a research grant from the National Insti- 
tute of Neurological Disease and Blindness of the National Institutes of Health, U. S. 
Public Health Ser\ice. 

The technical assistance of Mrs. Margarete Markey in preparing the tissue cultures 
is gratefully acknowledged. 

249 



250 F. STEPHEN VOGEL 

formation about the pathogenesis of transitory and lethal cellular responses 
of neurons to ionizing radiation. 

Methods 

Most procedures employed in these studies have been described in detail 
in individual publications. 

Total body radiation was administered to 67 young, 2- to 4-year-old, 
male Macacus rhesus monkeys, in graded doses from 1.000 to 30,000 r from 
a Ba^^"-La^^" source, at 1,000 r per minute. Detailed morphologic examina- 
tions were made of all animals promptly after death (Haymaker ct al., 
1958). 

Gamma radiation, in total doses of 10,000 r, was administered at 1,000 r 
per minute from a Go''" somce to 48 young male Macacus rhesus monkeys, 
divided into 3 groups of 16 animals each. One group received radiation to 
the head with the body shielded ; another, to the body with the head 
shielded; and a third, to the entire animal. At periodic intervals up to 96 
hours later, pairs of animals from each group were killed (Vogel ct al., 
1958). 

Explants of cerebellar tissue from mice, 3 days old, were grown on slides 
with prepared media (Gillete and Findley, 1958) in Garrel flasks and 
Maximow chambers. Gamma radiation was administered in doses of 10,000 
r from a Go"" Ticker machine at 160.5 r per minute at a distance of 30 cm. 
Irradiated and nonirradiated cultures were examined periodically by the 
phase microscope, and tissues were remoxed from the cultmes, stained by 
hematoxylin and eosin, and examined by light microscopy. 

Young healthy mongrel dogs and adult white albino rabbits were exposed 
to 15,000 r of gamma radiation from a Go''" Ticker machine over the 
superior cerebellar region with a field 5 cm in anterior-posterior dimension 
and 7 cm across. The rate was 160 r per minute with a source to skin dis- 
tance of 30 cm and a half value layer of 1 1 mm of lead. Tissues were 
taken from the cerebellum of anesthetized animals, fixed immediately in 1% 
osmium tetroxide solution, and prepared by standard methods for electron 
microscopy (Vogel, 1959). 

Observations 

Morphologic Effects of Gamma Radiation on the Brain as viewed 
WITH THE Light Microscope 

Exposure of animals to massive doses ol ionizing radiation is followed 
promptly by conspicuous morphologic alterations in the brain and mesen- 
chyma, notably by a pyknosis of the cerebellar granule cells and inflamma- 



EFFECTS OF HIGH-DOSE RADIATION ON BRAIN 251 

tion of the cerebral blood vessels, leptomeninties, and choroid plexuses. 
These lesions were not evident in monkeys exposed to 1,000 r. They were 
present in some animals, but equixocally or in minimal intensities, after 
exposine to 2,500 r. They were found with increasing frecjuency and in- 
tensity after doses of 5,000 and 10,000 r. They showed slight increases with 
£;reater dosages up to 30,000 r. The severity of the lesions differed appreci- 
ably from animal to animal. e\en when ex]30sure and sur\i\al conditions 
were essentially identical. 

The cytologic chanoes were well established within 2 hours after radi- 
ation. They were dynamic, for their intensity increased rapidly within the 
next 8 to 24 hours and then regressed precipitously, the lesions being min- 
imal or absent 96 hours after exposine. 

The pathologic alterations were ol the same character and intt'nsity 
whether the head and body or only the head was irradiated. They did not 
occm- when the radiation was applied to the body with the head shielded, 
the findings pro\idecl e\idence that these cytologic responses were induced 
by the ionizing rays acting directly upon the intracranial tissues, neither 
being initiated nor enhanced by exposure of other regions of the body. 

Granule Cp:ll Change 

The morphologic appearance ot the affected cells was notably sin:iilar in 
monkeys and rabbits, being regularly characterized by a reduction in the 
diameter of the nucleus to as much as one-half the normal, with marked 
condensation of the intranuclear chromatic material. Narrow margins of 
basophilic cytoplasm were visible about some of the contracted nuclei, and 
these often stained deeply with pyronin. Up to 50''r of the granule cells of 
a single animal were severely altered: most others remained normal: few 
showed intermediate degrees of change. Usually the affected cells were 
haphazardly distributed throughout all portions of the internal granular 
layer and cerebellum. In some animals, there was preferential localization, 
the vermis and deeper portions of the granular layer being most often so 
inxolved. Golgi and Purkinje cells were regularly spared. The pyknotic cells 
were more widely separated from one another than nonpyknotic ones. The 
appearance resembled that caused by extracellular edema, although regu- 
larly unaccompanied by coagulated fluid. The widened intercellular spaces 
were infiltrated by only a few leucocytes and macrophages and rarely con- 
tained hemorrhages. Neuronophagia was absent. Perivascular cuffing by 
leukocytes was minimal in the cerebellar cortex, particularly so in the 
granular layer. 

The initial cytologic changes in the granule cells of dogs was also charac- 
terized by contraction of the nucleus, but this was accompanied by nuclear 



252 



F. STEPHEN VOGEL 



' ^mtit 



%'t'i 



Fig. la. Granular layer of the cerebellum of a normal dog. The granule cells have 
uniform, rounded nuclei with a nucleolus and fine chromatin material, but no visible 
cytoplasm. Hematoxylin and eosin stain. X 400. 










Fig. lb. Granular layer of a dog 15 hours after exposure of the head to 15,000 r 
of gamma radiation from a Co'" source. Many nuclei are shrunken and hypcrchro- 
matic. Some show karyorrhexis. Hematoxylin and eosin stain. X 400. 



EFFECTS OF HIGH-DOSE RADIATION ON BRAIN 



253 



fragmentation in some (Figs, la, b). Macrophages, with phagocytized cellu- 
lar debris, were present. Notable losses of granule cells became evident in 
animals killed 8 days after exposure, and a mild astrocytic proliferation was 
present at this time. Golgi and Purkinje cells remained intact i Fig. 2). 

Vasculitis 

An exudate of leucocytes appeared promptly in all layers of the cerebral 
blood vessels. Veins and arteries were invoked about equally. Vessels of all 
sizes were affected. The vessels in the cerebral nuclear masses were generally 
more intensely involved than those in the cerebral cortex, while those in the 
white matter shared in the process, but to lesser degree. The vessels of the 
brain stem, cerebellum, and spinal cord were similarly affected, also in lesser 
intensity. The leucocytes rarely penetrated into the surrounding neural sub- 
stance, but usually concentrated in the ad\entitia and perivascular spaces. 
Hemorrhage was rare. Vessels stained specifically for collagen and elastic 
tissue regularly showed no notable alterations in these components. With 







Fig. 2. The rarified granular layer of a dog 10 days after exposure to lo.OOO r of 
gamma radiation contains CJolgi cells and an increased number of astrocytes, but is 
largely devoid of granule cells. Hemato.xyiin and eosin stain. X 55. 



254 F. STEPHEN VOGEL 

the passage of 72 to 96 hours, the inflammatory cells lysed and the exudate 
lessened, but with residual perithelial edema and scant numbers of macro- 
phages and lymphocytes still present at the latter times. 

Meningitis 

Initially the exudate was perivascular and spotty. It persisted as such in 
some animals, but disseminated over the gyri and spread into the sulci in 
most. The cellular composition varied with duration after exposure, but 
also difTered somewhat in animals with identical postirradiation states. 
Earlier polymorphonuclear leucoctyes predominated in great numbers; later, 
with decreases in the cellular concentrations, lymphocytes and macrophages 
were relatively more abimdant. Generally, macrophages persisted and with 
their contents of cellular debris constituted the inflammatory residue in 
animals killed 96 hours after exposure. 

Choroid Plexitis 

The choroid plexuses in the lateral, 3rd, and 4th ventricles were equally 
involved. The intensity and cellular composition of the inflammatory exu- 
date generally paralleled that in the meninges and in the cerebral blood 
vessels. Edema of the fibrous tissue stroma usually antedated the exudation 
of cells into these regions. The choroidal epithelium was generally spared, 
but ulceration followed on frons with imusually heavy exudates. The epi- 
thelium was reconstituted, and the inflammation had regressed in most 
animals by 96 hoius after exposure. 

Morphologic Effects of Gamma Radiation on Granule Cells in 

Tissue Culture 

The cells in explants of cerebellum from new born mice proliferated 
rapidly and migrated in sheets centrifugally onto the glass. Many of these 
cells had rounded nuclei with one or several small nucleoli, finely particulate 
chromatin material, a well defined nuclear membrane, and scant or un- 
detectable cytoplasm. Slender, short processes radiated from the perikaryon 
of some. These cells with distinctive cytologic features as .seen with the 
phase microscope and in sections stained with hematoxylin and eosin were 
considered granule cells (Fig. 3). Also abundant in cultures 5 to 10 days 
old were cells with more elongated nuclei, coarse chromatin material, and 
bipolar or diffusely radiating cytoplasmic strands. These resembled fibro- 
blasts derived from other tissue sources and were identified as such with 
phase and light microscopy. Some with similar appearances were viewed as 



EFFECTS OF HIGH-DOSE RADIATION OX BRAIN 



255 




Fig. 3. Granule cells of the cerelH-Uuin of a 3-day-old mouse grown for lU days in 
tissue rulturc. The cells ha\c rounded nuclei with extremely scant perikaryon. Phase 
Microscopy. X 1200. 



astrocytes. Fewer cells had a lars^er nucleus, usually with a prominent 
nucleolus, and abimdant perikaryon that formed a sinole dominant process 
and occasionally lesser ones. These cells were identified as neurons derived 
from the cerebellar cortex from regions other than the s;ranular layer. 

Preliminary studies have made it clear that only cells with the cytologic 
characteristics attributed to the granule cells showed notable structural 
changes in the immediate postirradiation period. These changes closely 
resembled those noted pre\iously in histologic preparations of the irradiated 
cerebellar cortex, being characterized by a contraction of the nuclei to 
approximately ~/i normal size. The perikaryon that was normally scant 
about the granide cells in tissue culture became conspicuously wnder and 
the over-all dimensions of many cells increased (Fig. 4). As noted in sections 
stained by hematoxylin and eosin. the nuclei were contracted and the 
chromatin material compressed and hyperchromatic. The perikaryon was 
more abimdant than normal, stained intensely with eosin, and it was often 
foamy and \acuolated. Altered cells were most abimdant 24 hours after 
exposuie. Periodic examinations ot the cultures with the phase microscope 



256 



F. STEPHEN VOGEL 




Fig. 4. Granule tells, as in Fig. 3, 24 hours after exposure to 10,000 r of gamma 
radiation from a Co"" source. The nuclei are markedly contracted and the nuclear 
membranes are serrated. The cytoplasmic spaces arc enlarged. The over-all size of 
these cells is somewhat less than normal; in others, it was greater. Phase Microscopy. 
X 1200. 

and in stained sections made it clear that these cytologic changes were 
transitory. The cells in individual clumps survived and became normal in 
appearance. Cells in nonirradiated cultures did not undergo these cytologic 
changes. 



Morphologic Effects of Radiation on Granule Cells as Viewed with 

Electron Microscope 

The fine structure of nonirradiated granule cells of rabbits was indis- 
tinguishable from that of the cells in dogs, and in each species they were 
strikingly uniform. The cells possessed a large, spherical nucleus with uni- 
formly distributed, finely granular, abundant intranuclear granules. The 
dual nuclear membranes were uninterrupted and lay parallel except for a 
rare out-folding of the external one. The cytoplasm was regularly scant, but 
clearly visible about the entire nucleus with expansions at the axon hillock. 



EFFECTS OF rilGH-DOSE RADIATION ON BRAIN 



257 



Mitochondria were small and sparse, rarely more than 6 in a single cross 
section of a cell. Their cristae were delicate and inconspicuous. The endo- 
plasmic reticulum was also scant, but was most abimdant at the axon hillock. 
A Golgi apparatus frequently occupied this region. Many granule cells lay 
side by side with cytoplasmic membranes in apposition. Others were encased 
in part or totally by dendritic processes (Fig. 5). 

The earliest recognizable cytologic changes, abundantly evident in the 
tissues of rabbits examined 24 hours after exposme, were characterized by 
a contraction of the nucleus and clumping of the intranuclear granules, 
with increased serration of the nuclear membranes and broadening of the 
cytoplasmic space with dispersion of the cioplasmir constituents. With 










■^^ ;* 

















Fig. 5. \ normal granule cell of a rabbit is encasrd in dendrites and has scant 
cytoplasm ( f ) with few mitochondria and sparse endoplasmic reticulum, .\pproxi- 
mately X 18.000. 



258 



F. STEPHEN VOGEL 



further contraction of the nuclei, the intranuclear material was greatly 
compacted, but still remained particulate and discrete, the granules being 
separated from one another by an electron-lucent margin of uniform width. 
Although the nuclear membranes became redundant with extreme contrac- 
tion of the nuclear mass, in the rabbit they did not fragment, but folded 
and coiled. The expansion of the cytoplasm was conspicuous, in most cells 
exceeding the volumetric decrease in the nucleus. The over-all size of the 
cells increased. The endoplasmic reticulum was widely dispersed in the 
expanded cytoplasm, but did not show consistent structural change (Fig. 6). 
The recovery phase, as judged by light microscopy, was completed by 72 
hours after radiation. Tissues taken at this time and examined with the 




Fig. 6. Electron micrograph of a pyknotic granule cell of a rabbit 24 hours after 
exposure to 15,000 r of gan.ma radiation. The nucleus of one cell is extremely con- 
tracted; the intranuclear granules are condensed and the cytoplasmic space ( f ) is 
greatly expanded, with dispersion of the cytoplasmic constituents. Two unaltered 
granule cells and a capillary surround the pyknotic one. Approximately X 10,000. 



EFFECTS OF HIGH-DOSE RADIATION ON BRAIN 



259 




Fig. 7. Electron micrograph of karyorrectic granule cell of a dog 24 hours after 
exposure to 15,000 r of gamma radiation. There is fragmentation of the nuclear mem- 
brane and extrusion of the cytoplasmic constituents. Approximately X 15.000. 

electron microscope showed most of the lirannle ceUs to ha\e fine structure 
that was essentially normal. Minor stigmata were present in some, indenta- 
tions of the nuclear membranes bein^ the most conspicuous. 

In general, the altered neurons in radiated dogs were not notably different 
from those in rabbits. The initial cytologic changes were, as in rabbits, shink- 
age of the nuclei, clumping of the nuclear granules, folding and redundancy 
of the nuclear membranes, and expansion of the cytoplasm. In addition, and 
apparently as a further progression of these changes, there was fragmentation 
and disintegration of the nuclear membranes of some of the contracted 
nuclei (Fig. 7). 

Comment 



The findings make it clear that exposiue of the granule cells in the intact 
animal or in tissue culture to ionizing radiation initiates rapid volumetric 



260 F. STEPHEN VOGEL 

changes in the intracelkilar compartments. It seems most likely that these 
changes are manifestations of a rapid transferal of electron-lucent "nuclear 
sap" from the karyoplasm into the perikaryon. Such volumetric changes 
might result from alterations in the permeability of the nuclear membranes, 
from hypotonicity in the nuclear chamber, which causes an egress of fluid, 
from hypertonicity in the cytoplasmic compartment, which attracts fluid, or 
from several of these factors acting simultaneously. The expansion of the 
cytoplasmic space, which in many cells notably exceeded the volumetric 
decrease in the nucleus, and the increase in the over-all size of the cells make 
it seem likely that extracellular fluid is also imbibed by the perikaryon. Con- 
sidered together, the findings suggest that radiation induces a marked hyper- 
tonicity of the cytoplasm that, in turn, initiates a series of cytologic changes 
that are transitory in the granule cell neurons of rabbits and other animals, 
but often lead to cellular death in those of the dog. 

References 

Alvord, E. C, and Brace, K. C. 1957. X-ray induced pyknosis of cerebellar granule 
cells in guinea pigs and its suppression by barbiturate anesthesia. /. Neuropathol. 
Exptl. Neurol. 16, 3. 

.Arnold, A., Bailey, P., and Harvey, R. A. 1954. Intolerance of the primate brain stem 
and hypothalamus to conventional and high energy radiation. Neurol. 4, 575. 

Briinner, H. 1920. l)ber den Einfluss der Rontgenstrahlen auf das Gehirn. Arch. klin. 
Chir. Langenbecks 114, 332. 

Gerstner, H. B., Brooks, P M., Vogel, F. S., and Smith S. A. 1956. Effects of head 
x-irradiation in rabbits ( <\ aortic blood pressure, brain water content, and cerebral 
histology. Radiation Rest rch 5, 318. 

Gillete, R., and Findley, .A, 1958. A simple technique for studying the effects of cul- 
tivation in vitro on skin grafts. Transplantation Bull. 5, 124. 

Haymaker, W., Nauta, W. J. H., Sloper, J. C., Laqueur, G. L., Pickering, J. E., and 
Vogel, F. S. 1958. The effacts of Barium'*" — Lanthanum"" (Gamma) radiation on 
the central nervous system and pituitary gland of Macaque monkeys. /. Neuropathol. 
Exptl. Neurol. 17, 12. 

Hicks, S. P., and Wright, K. A. 1954. Variation of pathological responses to radiation 
with time intensity factors. Am. ] . Clin. Pathol. 24, 77. 

Vogel, F. S. 1959. Changes in the fine structure of cerebellar neurons following ioniz- 
ing radiation. /. Neuropathol. Exptl. Neurol. 18, 580. 

Vogel, F. S., Hoak, C. G., Sloper, J. C., and Haymaker, W. 1958. The induction of 
acute morphological changes in the central nervous system and pituitary body of 
Macaque monkeys by Cobalt"" (Gamma) radiation. /. Neuropathol. Exptl. Neurol. 
17, 138. 

Wilson, S. G. 1960. Radiation-induced central nervous system death. A study of the 
pathologic findings in monkeys irradiated with massive doses of Cobalt"" (Gamma) 
radiation. /. Neuropathol. Exptl. Neurol. 19, 195. 



Electron Microscope Observations on the 

X-lrradiated Central Nervous System of the 

Syrian Hamster* 

H. Hager, W. Hirschberger, and A. Breit 

Deutsche Forschungsanstalt fur Psychiatrie 
Max-Planck-Institut, Munich, Germany 



Relathely few reports on brain damase follovvinsj massive dosages of 
x-radiation have been published in the light microscope literature (Hicks 
and Montgomery. 1952: Hicks et al., 1956). and only Vogel (1959) has 
utilized the high resolving power of the electron microscope in his study of 
x-ray induced pyknosis of the granular cells of the cerebellum. Our work 
was imdertaken to demonstrate the morphologic alterations produced by 
x-radiation and to ascertain if there exist in the ultrastructural range cyto- 
logic alterations specific for this type of energy. The brains from which speci- 
mens were removed for electron microscopy were also utilized in the exten- 
sive light microscope investigations of Scholz which are reported elsewhere 
in this symposium. Only ultrastructural observations will be considered. 
Because of the sampling limitations inherent in electron microscope tech- 
nique, time-dosage relationships will not be discussed. 

Material and Methods 

A circumscribed region ol the mediodorsal skull of unanesthetized Syrian 
hamsters was subjected to a single exposure of .x-radiation. A Monophos 
ray machine was used with a target distance of 5.5 cm. The technical con- 
ditions were as follows: 40 KV, 25 niA. 0.3 Aluminum with a half value 
depth in tissue of 1 mm. Dosages ranged from 7,500 to 45,000 r, and animals 
were sacrificed after periods ranging from 4 hours to 30 days. Specimens 
taken from the living animal were fixed in buffered osmium tetroxide, 

* This project was supported by the School of .\viation Medicine of the .•\ir Re- 
search and Deselopment Command, U. S. .Air Force, through its European Office. 

The authors express their deep appreciation to Dr. Erland Nelson, University of 
Minnesota, for his heljjful suggestions and assistance in the preparation of this man- 
uscript. 

261 



262 



H. HAGER, W. HIRSCHBERGER AND A. BREIT 



treated in the usual fashion, and embedded in methacrylate. Uhrathin 
sections were examined in a Siemens t)M 100 electron microscope/ 

Results and Discussion 

This preliminary report is concerned only with dosages sufficiently high 
to produce necrosis. 

Within a few hours after exposure to the radiation the first significant 
change that could be seen with the electron microscope consisted of swelling 

'"MA 







AP 




'N' 












Fig. 1. Cerebral cortex, 15 hr after exposure to 15,000 r. Swelling of astrocytic 
processes (AP) in the neuropil and around a capillary (C). N, nerve cells. X 4,250. 

^ The authors gratefully acknowledge the techincal assistance of Miss Luh and Mr. 
Fellner and the cooperation of Professor Dr. Rollwagen in allowing the use of the 
electron microscope facilities of the University of Munich. 



X-IRRADIATED CNS OF THE SYRIAN HAMSTER 



263 



of the astrocytes" cytoplasm i Fig. 1 ) . All astrocytic processes are considerably 
enlarged and even more pale and "watery" than usual. Around a small 
capillary there is disruption of astrocytic membranes, probably due to a 
combination of artifact and edema. Under different experimental conditions 
De Robertis et al. 1958 ) have produced similar changes and emphasized the 
role of the astrocytes in water and ion metabolism of the brain tissue. It 
seems probable that this astrocytic swelling represents the electron micro- 
scope equivalent of the sponginess and loosening of the cortical neuropil that 
is usually considered reflective of edema. This morphologic manifestation of 
radiation-induced edema is restricted to astrocytic cytoplasm, and other cells 
within the central nervous system seem to be unaffected. Furthermore, the 
narrow intercellular gap which represents the only anatomic extracellular 
space in the brain i Hager. 1959: Horstmann and Meves. 1959) is not in- 
creased in this situation. The neurons appear normal at this stage. The 
appearance of mitochondria is somewhat difficult to evaluate, since similar 
changes in these organelles can occur as the result of incomplete or delayed 
fixation. 

Figure 2 demonstrates erythrodiapedesis around a small \ein. The red 
blood cells lie outside the perivascular space and within the intercellular gap. 
This is more easily seen in Fig. 3. where the erythrocyte is situated between 
completely intact cell processes, most of which are astrocytic. Contemporarily 
with the appearance of astrocytic swelling and erythrodiapedetic bleeding, 
there occurs slight, but probably definite, swelling of the capillary endo- 




l"iu. -. Ucicbial cortfx. 69 hr after exposure to 45,UUU i. Ei\ ihiodiapcdcsis around 
a small vein outside of the perivascular space. E, erythrocytes lying between astrocytic 
processes. V, vessel wall; N. nen.e cell. X 4,800. 



264 



H. HAGER, VV. HIRSCHBERGER AND A. BREIT 




Fig. 3. Cerebral cortex, 48 hr after exposure to 45,000 r. A diapedetic erythrocyte 
(E) lies between completely intact cell processes in the neighborhood of a capillary. 
EC, swollen endothelial cytoplasm; EN, endothelial nucleus; O, outpouching in the 
capillary lumen; B, basement membrane of capillary. X 10,800. 



thelium. Occasional outpouchings and irregularities in the capillary lumen 
can also be seen. Cytoplasmic organelles and vascular basement membranes 
show no significant alterations. 

Similar changes in the blood vessels and remarkable edematous astrocytes 
are also noted in irradiated cerebellar tissue (Fig. 4). Here, however, nerve 
cells are also involved, though there is a striking difference in the appearance 
of granular cells and Purkinje cells. The latter are relatively unchanged, 
while the granular cells are shrunken and hyperchromatic with clumping of 
the nuclear material. A higher magnification of such changes is seen in 
Fig. 5, where normal and abnormal granular cell nuclei can be contrasted. 
These pyknotic changes in granular cells following radiation have been 
recognized by several investigators (Alvord and Brace, 1957; Briinner, 1920; 
Haymaker ct ai, 1954, 1958; Schummelfeder, 1957: Vogel et al., 1958), and 
similar electron micrographs have been published by Vogel (1959). 

In Fig. 6 the extensive plasma exudation following radiation is apparent. 
The perivascular space bounded by the glial and vascular basement mem- 
branes (Nelson <7 ai, 1961 ) is filled with structureless material, which has 
a density similar to that of plasma and contains inflammatory cells. Neither 
leucocytes nor erythrocytes have so far been seen penetrating the glial base- 
ment membrane that constitutes the external margin of the perivascular space, 



y^ -* " ^A^'^' 














^ t, 






- / 



4 vv 



,* > 



Er-T ^^^.'V "l* -V. ^ 



♦ *!- s. 

Bgl* 



m , 







-*,' 4-. 






Fig. 4. Cerebellar cortex, Purkinje cell layer and superficial granular layer, 22 hr 
after exposure to 40,200 r. Scattered pyknosis of granular cells (PG). NG, normal 
granular cell: Pu, perikaryon of a Purkinje cell; Bgl, Bergmann glial cell; Bgls, Swell- 
ing of Bergmann gliocytic processes. X 4.800. 




Fig. 5. Cerebellar cortex, granular layer, 46 hr after exposure to 30,000 r. Nuclei 
of granular cells show severe shrinkage and clumping of the nucleoplasm (PG). NG, 
normal granular cell nucleus. X 10,800. 

265 



266 



H. HAGER, W. HIRSCHBERGER AND A. BREIT 







.k \\n- Ak 






.gbM 





•>^. 



Fig. 6. Cerebral cortex, 45 hr after exposure to 15,000 r. Tangential section through 
a small vein. The perivascular space (PVS), bordered by glial (GBM) and vascular 
(VBM) basement membranes, is filled with exudate and also contains a monocyte 
(Mo). Ex, exudate within partially necrotic tissue; Er, diapedetic erythrocytes; As, 
astrocytic processes containing densely osmiophilic inclusions. X 4,500. 



though polymorphonuclear leucocytes have been literally caught in the act 
of passing through the vascular basement membrane. This process has been 
discussed elsewhere (Nelson et al., 1960), but we are still ignorant of the 
factors that prevent or permit such cellular movement. Plasma exudate is 
also seen outside the confines of the perivascular space, but it is difficult to 
determine whether this is in the intercellular space, as are the erythrocytes, 
or if it is partly intercellular in these incompletely necrotic tissue. A rather 
common finding is the occurrence of densely osmiophilic inclusions in astro- 
cytic cytoplasm. Figure 7 shows exudate associated with more definite early 
necrosis, with dissociation of the normal, intricately arranged components of 
the neuropil and a disruption of some of their membranes. Nerve cells, such 



X-IRRADIATED CNS OF THE SYRIAN HAMSTER 



267 




Fig. 7. Cerebral cortex, 73 hr after exposure to 45,000 r. Beginning necrosis with 
dissociation of the components of neuropil and disruption of some of their membranes. 
Nerve cell (N) shows widening of the endoplasmic reticulum and swelling of mito- 
chondria. Ex, exudate. X 4.000. 



as the one shown here, contain a nucleus which morpholooically is still essen- 
tially normal, while the cytoplasm shows changes of questionable significance, 
including widening of endoplasmic reticulum and mitochondrial swelling. 
There is good preservation of RNA granules. 

In Fig. 8 one can see a still more severe stage of tissue destruction. Here, 
a small artery is in a state of relatively good preser\ation. despite extensi\e 
necrosis of the surrounding tissue. The outer margin of the perivascular space 
is no longer intact. Perivascular macrophages and leucocytes are seen in 
contact with the necrotic neuropil. The dark round bodies are probably 
ingested fragments of erythrocytes. 

Figure 9 illustrates total dissociation of tissue components so that only cell 



268 



H. HAGER, W. HIRSCHBERGER AND A. BREIT 



PMN 



AR 





^^^■■■^^m^,^ 



•V^^" 
f^-^-" 

^ 



<: -0 '-'- ■ ■ ^ ^-^ 'fl^^ 







0*. ■*» 









/^: 



.-^Z 



..s.^' 






,M 



EF 



.i 



Fig. 8. Cerebral cortex, 73 hr survival after exposure to 45,000 r, showing a rela- 
tively well preserved small artery (Ar) surrounded by macrophages (M) and poly- 
morphonuclear leucocyte (PMN) in an area of extensive necrosis. EF, ingested ery- 
throcyte fragments. X 4,000. 



membranes and occasional mitochondria are still recognizable. Erythrocytes 
and macrophages are seen swimming free in the severely damaged tissue. 

Figure 10 is a high magnification of a siinilar region to show the variety 
of intracytoplasmic inclusions in macrophages. The significance and origin 
of similar products of decomposition in cerebral necrosis following me- 
chanical trauma has already been reported. fHager, 1960). In the same 
figure, the persistent preservation of membranous components of the neuropil 
is evident. Figure 1 1 shows a nerve cell also in a severe stage of destruction. 
Nuclear and cytoplasmic membranes are interrupted. The internal arrange- 
ment of the mitochondria is broken down, and the normal fine structural 
organization of the cytoplasm is greatly altered. The nucleoplasm is vacu- 
olated, no longer fills the nuclear envelopes, and is obviously abnormal, 
though the appearance differs greatly from the shrunken pyknotic nuclei of 
the granular cells of the cerebellum. Capillaries from areas of total necrosis. 







^,:, 



■-^" -*«'■ 






■<Jt ' 



A 
















^" 



Fig. 9. Cerebral cortex, 172 hr after exposure to 7,50U r. Fully developed necrosis 
with total dissociation of most tissue components, though cell membranes are still 
recognizable. Macrophages (M) and erythrocytes (Er) arc seen free in the necrotic 
tissue. X 4,000. 



^: 




•• 




"■.^ , .. 


M- 


' - -^; 


^ ^ 


^^ 


-^£r 


^^m^ 


;ef;^ 


^ 


t 

:-4 




Fig. 10. C^iirljial cuitr.x, 172 hr after exposure to 7,500 r. Nancus tytoplasmic 
inclusions in a macrophage (M). EF, erythrocyte fragments; MyF, myelin figures; 
Fe, hematogenic pigment, probably iron-containing; CM, cell membranes of dissociated 
components of the neurophil; Mi, mitochondria. X 10,800. 



269 



270 H. HAGER, W. HIRSCHBERGER AND A. BREIT 




Mi; : • ■ . ,:-= cp 

-^ r ^ . . . ' . • . • • ^ ^ V -' 

^ ' \. ' ! . -'' X CM 

• .> • 




J. • ^ 



y 



Fig. 11. Cerebral cortex, 172 hr after exposure to 7,500 r. Interruption of nuclear 
(NM) and cytoplasmic (CM) membranes of a nerve cell with breakdown of internal 
arrangement of mitochondria (Mi). Severe changes in appearance and distribution of 
nucleoplasm (Np) and cytoplasm (Cp). X 21,600. 

as in Fig. 12, are also strikingly alteied. One can recognize enormous swell- 
ing of the endothelial cytoplasm, vaciiolation, and various types of inclusion 
bodies, so that these cells appear to have a phagocytic function. The base- 
ment membrane remains relatively well preserved. In Fig. 13, at the border 
zone of a necrotic area, two large reactive astrocytes are seen. The cytoplasm 
of these cells is clearly of the same type as the pale, watery cytoplasm that 
previously has been considered characteristic for astrocytes (Farquhar and 
Hartmann, 1957; Schultz et ai, 1957). The presence of intracytoplasmic 
fibrils in these cells, which can be seen in Fig. 14 at higher magnification, 
would seem to be a significant point in the positive identification of this cell 
type as an astrocyte and not as an oligodendrocyte as has been maintained 
by some investigators (Briinner, 1920; Luse, 1958). 



X-IRRADIATED CNS OF THE SYRIAN HAMSTER 271 









V 







Fig. 12. Cen-bral cortex, 168 hr after exposure to 7.500 r showing alterations in a 
capillary in an area of total necrosis. Basement membrane (BM) is still preserved, 
while endothelial cytoplasm is swollen and contains vacuoles (V), myelin figures 
(MyF), and various other types of inclusions. Er, erythrocytes. X 10,800. 



Conclu-sions 

The early alterations consisting of astrocytic swelling, plasma exudation, 
and erythrodiapedesis are all to be considered secondary to increased vascular 
permeability. It would seem that an acute effect of ionizing radiation is to 
interfere with basic cellular mechanisms of the cell and. while probably 
affecting all elements of the central nervous system, to have its most dramatic 
effect on the blood \essels which manifest this functional derangement by 
increased permeability, thus allowing fluid and erythrocytes to pass. Changes 
in the granular cells of the cerebellum similar to those lollowing x-irradia- 
tion ha\e been described in a number of conditions where there was definite 
or presimiptive e\idence of edema 1 Leigh and Meyer, 1949: Notzel, 1955: 
Olsen, 1959a, b; Schmidt, 1958: Upners. I939'i. It is. therefore, entirely 
possible that the striking change of granidar cells is not a direct radiation 
effect, but is a nonspecific reaction of this type of cell to the presence of 
edema. A study on ultrastructural alterations in the brain following extensive 
cerebral trauma has been made in oiu" laboratory, parallel with investigations 
on the effect of x-ray-induced damage Hager, 1960). These different, but 
complementary, studies can profitably be compared when seeking cytologic 



272 



H. HAGER, W. HIRSCHBERGER AND A. BREIT 











^ 









} 









'■"¥. 



» 









/'♦ 



* 






^ 



,-kJtf^'-a 



La 






Mi 



Osi 



Ml 





Osi 


^rS. '^H'- 


Mi 


■5» fv « 


Fi 



Fi 






Osi 



Fig. 13. Cerebral cortex, 218 hr after exposure to 7,500 r. Large reactive astrocytes 
(As) in the border zone of a necrotic area. Intracytoplasmic fibrils (Fi) can be seen 
in the perinuclear regions. Osi, osmiophilic inclusions; Mi, mitochondria. X 4,000. 



X-IRRADIATED CNS OF THE SYRIAN HAMSTER 



273 




Fig. 14. Cerebral cortex, 218 hr after exposure to 7,500 r showing perinuclear 
region of a reactive astrocyte. The cytoplasm contains many fine fibrils (Fi). N, nu- 
cleus. X 21,600. 



alterations that might be specific for ionizing radiation. With our present 
knowledge, it is not possible to be dogmatic about the significance of observa- 
tions which, though the result of examining many himdreds of microphoto- 
graphs, still are based on alterations in only a relatively small number of cells 
from limited areas. Nonetheless, it appears that in areas of necrosis following 
x-radiation there is a depression of phagocytic elements in number and 
activity. Thus, from areas of total traumatic necrosis, the macrophages are 
filled with vacuoles and inclusions, while in regions of correspondingly com- 
plete x-ray induced necrosis, the phagocytes aie fewer and their phagocytic 
function less prominent. 

It is possible that ionizing radiation affects the ability of macrophages to 
proliferate in and around a necrotic area, as well as altering the enzymes 
involved in the breakdown of phagocytosed material. 



274 H. HAGER, W. HIRSCHBERGER AND A. BREIT 

References 

Alvord, E. C, and Brace, K. C. 1957. X-ray induced pyknosis of cerebellar granule 

cells in guinea pigs, and its suppression by barbiturate anesthesia. /. Neuropathol. 

Exptl. Neurol. 16, 3-17. 
Briinner, H. 1920. Uber den Einfluss der Rontgenstrahlen auf das Gehirn. Arch. Klin. 

Chir., Langenbecks 114, 332. 
Dempsey, E. W., and Luse, S. A. 1958. Fine structure of the neuropil In relation to 

neuroglia cells. In "Biology of Neuroglia," pp. 99-108. Charles C Thomas, Spring- 
field, Illinois. 
De Robertis, E. D. P., Gerschenfeld, H. M., and Wald, F. 1958. Some aspects of glial 

function as revealed by electron microscopy. Proc. 4th Intern. Conf. on Electron 

Microscopy, Berlin, 1958 2, 443-447. 
Farquhar, M. G., and Hartmann, J. F. 1957. Neuroglial structure and relationships 

as revealed by electron microscopy. /. Neuropathol. Exptl. Neurol. 16, 18-39. 
Hager, H. 1959. Elektronenmikroskopische Untersuchungen iiber die Struktur der 

sogenannten Grundsubstanz in der Gross- und Kleinhirnrinde des Saugetieres. Arch. 

Psychiat. Nervenkrankh. 198, 574-600. 
Hager, H. 1960. Elektronenmikroskopische Befunde zur Cytopathologie der Abbau — 

und Abraumvorgange in experimentell erzcugten traumatischen Hirngewebsne- 

krosen. Naturwissenschaften in press. 
Haymaker, W., Laqueur, G. L., Nauta, W. J. H., Pickering, J. E., Sloper, J. C., and 

Vogel, F. S. 1958. The effects of Barlum-Lauthanum (Gamma) radiation on the 

central nervous system and pituitary gland of Macaque Monkeys. /. Neuropathol. 

Exptl. Neurol. 17, 12-57. 
Haymaker, W., Vogel, F. S., Cammermeyer, J., Laqueur, G. L., and Nauta, W. J. H. 

1954. Effects of high energy total-body gamma irradiation on the brain and pituitary 

gland of monkeys. Am. J. Clin. Pathol. 24, 70. 
Hicks, S. P., and Montgomery, P. O. B. 1952. Effects of acute radiation on the adult 

mammalian central nervous system. Proc. Soc. Exptl. Biol. Med. 80, 15. 
Hicks, S. P., Montgomery, P. O. B., and Leigh, K. E. 1956. Time-intensity factors in 

radiation response. 1. The acute effects of megavolt electrons (cathode rays) and 

high- and low-energy x-rays with special reference to the brain. A.M. A. Arch. 

Pathol. 61, 226. 
Horstmann, E., and Meves, H. 1959. Die Feinstruktur des molekularen Rindengraues 

und ihre physiologische Bedeutung. Z. Zellforsch. u. mikroskop. Anat. 1959, 49, 

569. 
Leigh, A. D., and Meyer, A. 1949. Degeneration of the granular layer of the cere- 
bellum. /. Neurol. Neurosurg. Psychiat. 12, 287-296. 
Luce, S. A. 1958. Ultrastructure of reactive and neoplastic astrocytes. Lab. Invest. 

7, 401-417. 
Nelson, E., Blinzinger, K. H., and Hager, H. 1961. Electron microscope observations 

on the subarachnoid and perivascular space in the brain of the Syrian hamster. 

Neurology 6, 285. 
Nelson, E., Blinzinger, K. H., and Hager, H. 1960. An electron microscope study 

of experimental meningitis. Manuscript in preparation. 
Notzel, H. 1955. Schadigung und Verkalkung der Kornerschlcht des Klelnhirns bei 

chronischer experimenteller Sublimat\'ergiftung. Beitr. pathol. Anat. u. allgem. 

Pathol. 115, 226-236. 



X-IRRADIATED C\S OF THE SYRIAN HAMSTER 275 

Olsen, S. 1959a. t)ber die akute Nekrose der Kornerschicht des KKinhirns. Arch. 

Psychiat. Nerrenkrankh. 199. 1-13. 
Olsen, S. 1959b. Acute selective necrosis of the granular layer of the cerebellar cor- 
tex. /. NeuTopathol. Exptl. Neurol. 18, 609-619. 
Schmidt, H. 1958. Die Bedeutung der Kornerschichtnekrose der Kleinhirnrinde fiir 

die histologischc Diagnose der Hirnschwellung. Verhandl. deut. Ges. Pathol. 41. 

241-246. 
Schultz, R. L., Maynard. E. A., and Pease. D. C. 1957. Electron microscopy of 

neurons and neuroglia of cerebral cortex and corpus callosum. Am. J. Anat. VI. 

100, 339-407. 
Schiimmelfeder, N. 1957. Fluoreszenzmikroskopische und cytochemische Unter- 

suchungen iiber Friihschaden am Kleinhirn der Maus nach Rontgenbestrahlung. 

/"'■ Congr. intern. Sci. Neurol., Brussels, 1957: 3' Congr. intern. Neuropathol. 

pp. 295-308. Acta Medica Belgica, Brussels. 
Upncrs, T. 1939. Experimen telle Untersuchungen iiber die lokale Einwirkung des 

Thiophens im Zentralner\ensystem. Z. ges. Neurol. Psychiat. 166. 623-645. 
Vogcl, F. S. 1959. Changes in the fine structure of cerebellar neurons following 

ionizing radiation. /. Neuropathol. Exptl. Neurol. 18, 580-589. 
Vogel, F. S., Hoak, C. G., Sloper. J. C, and Haymaker, W. 1958. The induction of 

acute morphological changes in the central nervous system and pituitary body of 

macaque monkeys by cobalt"" (gamma) radiation. /. Neuropathol. Exptl. Neurol. 

17, 138. 



Bioelectric Effects of High Energy 
Irradiation on Nerve* 



C. T. Gaffey 

Donner Laboratory, University of California, 
Berkeley, California 



Introduction 

There is little in the literature concerning the action of high energy radia- 
tion on the bioelectric properties of nerve. Most papers deal with the exposure 
of nerve to x-rays (Audiat, 1932; Audiat and PiflFault, 1934; Audiat, et al, 
1934; Bachofer, 1957; Bachofer and Gautereaux, 1959, 1960a, b; Gerstner, 
1956; Gerstner et al, 1955; Janzen and Warren, 1942) or beta rays (Gas- 
teiger, 1951, 1952, 1959; Redfield ct al, 1922). The goal of this investigation 
was to determine the dose of high energy radiation that would inhibit the 
excitatory process of frog's sciatic nerve. Synchrocyclotron-produced 910 
Mev alpha particles, and 455 Mev deuterons were employed as irradiation 
beams. 

The effects of high energy alpha particles and deuterons have medical 
implications because of the increasing application of cyclotron beams to 
stereotaxic radiosurgery in the central nervous system (Tobias et al, 1952, 
1958; Born et al, 1959). In space exploration and in long time exposure 
projects, such as lunar colonization, the biologic effects of high energy par- 
ticles might be a limiting factor. Evaluation of this hazard has been specula- 
tive. A practical way to study this problem is to engage existing cyclotron 
facilities for biologic research. 

Methods 

Frogs {Rana pipiens) were housed under low temperature conditions 
(10°C) for about a week prior to experimentation. They were sacrificed by 
decapitation followed by spinal cord pithing. Both sciatic nerves were excised 
from over 200 frogs and placed in Ringer's solution (Mitchell, 1948). One 

* This study is based on work performed under contracts with the U.S. Atomic 
Energy Commission. 

277 



278 



C. T. GAFFEY 



nerve of each pair was irradiated, while its companion functioned as a 
control. 

To determine neural activity, a nerve was placed on Ag-AgCl electrodes 
in a moist chamber (Fig. 1) through which circulated a mixture of 95% 
oxygen and 5% carbon dioxide saturated with water vapor after passage 
through 3 gas-washing cylinders. Monophasic, rectangular stimuli 0.1 milli- 
sec in duration were delivered from a Grass stimulator (Model S-5) through 
an isolation unit to the nerve at 60 pulses per second. Recording electrodes 
detecting the propagated neural impulse ran to a push-pull, A. C. pre- 
amplifier (Grass Model P-5), which then fed the signal into a Textronic 
oscilloscope (Model 532) with a high-gain, differential input amplifier (Tex- 
tronic type 53/54 D). In conduction velocity studies a fast rise, dual-trace 
input stage amplifier (Textronic type 53/54 C) was employed. The dis- 
played action potentials were photographed by a Fairchild polaroid oscillo- 
scope camera (Model F-286"). 

The Lawrence Radiation Laboratory's 184-in. frequency modulated cyclo- 
tron was available as a source of 910 Mev alpha particles and 455 Mev deu- 
terons (Tobias et al, 1952, 1958). By appropriate magnetic focusing tech- 
niques, these high energy nuclei were made to travel in parallel, approxi- 
mately monoenergetic beams. An ionizing chamber placed in front of the 
bombarded nerve was used to monitor the delixered dose (Birge et al., 
1956). A summary of the specifications of the 184-in. synchrocyclotron is 
presented in Table L 



\ Outflow 




Electrodes 



Inflow of water saturated 
95% Og + 5% COg 

Fig. 1. Lucite chamber for keeping a nerve moist during the study of propagated 
potentials. Electrodes (1) and (2) are stimulating electrodes, while recordings can 
be made from electrodes (3) and (5) or (4) and (5). The distances from the first 
electrode are: 3 mm, 15 mm, 30 mm, and 40 mm. 



460 


915 


0.75 


0.10 


120 


16 


4.5 


4.5 


110,000 


110,000 


550 


550 


0.59 


0.59 


24 


24 


12 


7 


44 


22 



HIGH ENERGY IRRADIATION: BIOELECTRIC EFFECTS 279 

T.\BLE I 
Summary of Specifications of the 184-inch Synchrocyclotron 

Beam particles Deuterons Alpha particles 

Beam energy — maximum (Mev) 

Beam intensity — average current (ua) 

Beam intensity — peak current (ua) 

Time required for acceleration (msec) 

Number of revolutions during acceleration 

Distance traveled during acceleration (miles) 

Velocity at maximum energy (v/c) 

Mass increase at maximum energy {% of rest mass) 

Range of particles (in. of Al) 

Range of particles (gm/cm? of tissue) 

Under most experimental conditions, the dose rate received by nerves was 
2 krad per min (1 krad = 10^ ergs absorbed per gm or 1.07 X 10^ rep 
absorbed in tissue). High energy nuclei were generated by the 184-in. syn- 
chrocyclotron in pulses of 500 microsec duration with 64 pulses per sec. In 
special experiments the effect of varying the dose rate of the cyclotron's 
beam from 0.5 to 8.0 krad per minute was tested to determine if this was 
a significant factor in altering neural activity. The linear energy transfer 
(also referred to as stopping power and rate of energy loss) of alpha parti- 
cles was 15 Mev-cm- per gm (Bom et al., 1959), i.e., approximately the 
same linear energy transfer of secondary electrons from a 250 kev x-ray 
machine. 

Results 

Bioelectric Studies 

In exploratory experiments nerves mounted in a moist chamber were 
placed in the horizontal path of high energy particles generated by the 
184-in. synchrocyclotron. Irradiation of the nerve was beyond the stimulating 
electrodes (maximum beam diameter was 44 mm). Every 10 krad, the cyclo- 
tron's beam was interrupted, and the action potential of the nerve being 
irradiated was recorded photographically until the electrical activity was 
abolished. Large doses of alpha particles were required to block excitation. 
It is now known that there is a serious difficulty with this type of procedure 
because a greater dose than minimal was received by the nerve to eliminate 
its electrophysiologic response. 

To determine the effect of high energy particles on neural activity, it was 
deemed prudent to follow the time course of the survival of bioelectric activ- 



280 C. T. GAFFEY 

ity after exposure to some specified dose of irradiation. For this purpose the 
following method was adopted. After obtaining oscillograms of the preirradi- 
ated neural activity of both isolated sciatic nerves of a frog, one nerve of the 
pair was bombarded in the cyclotron's beam while contained in a plastic 
vial filled with Ringer's solution. Following irradiation, the neural activities 
of the exposed and control nerve were again monitored after transferral to 
a moist chamber (Fig. 1). This routine was repeated at 2 hour intervals for 
a minimum of 24 hours. Control nerves maintained in Ringer-filled vials 
were treated in an identical manner. 

In Fig. 2 are shown three rows of oscillograms of the action potentials of 
the right (upper photographs) and left (lower photographs) sciatic nerves 
of a frog. Preirradiation action potentials were recorded, and the right sciatic 
nerve was subjected to 72 krad of 910 Mev alpha particles. The left sciatic 
nerve functioned as a control. Immediately after alpha particle irradiation 
(oscillograms above "0 hr" in Fig 2), a transformation in the action poten- 
tial complex of the exposed nerve was apparent. Oscillograms recorded at 2, 
4, 6, 8, 10, and 12 hours after irradiation trace the deleterious effects caused 
by alpha particles. At 14 hours postirradiation, there was complete cessation 
of the bioelectric activity of the alpha-bombarded nerve, while the action 
potential of the control nerve was still present. 

The spike potential changes for the irradiated and control nerve illustrated 
in Fig. 2 are summarized in Fig. 3. On the ordinate of Fig. 3 (and also on 
the ordinates of Figs. 4, 5, and 6) is plotted the percentage of the initial spike 
potential, i.e., the ratio of the amplitude of the spike potential at some t-hours 
after irradiation over the preirradiated spike potential amplitude multiplied 
by 100. 

In Fig. 4 is presented a sample of the data obtained for alterations in the 
neural activity resulting from alpha particle irradiation. It is clear that larger 
doses of alpha particles (greater than 300 krad) eliminate neural excitability 
rapidly. With lower doses of 910 Mev alpha particle irradiation, the survival 
of neural activity is progressively extended. It would appear from Fig. 4 that 
at 6 hours postirradiation there is considerable enhancement of the neural 
output. That all this enhancement is a direct consequence of irradiation 
seems doubtful, because when the irradiated nerve of a pair demonstrated 
an enhanced neural output, so did its nonirradiated control (Fig. 5). How- 
ever, bombarded nerves with enhanced activity were usually 5 to 10% higher 
in their neural output than their controls. The nonirradiated nerves mani- 
fested the enhancement phenomena most strongly during the winter season. 

The time course for the abolition of neural activity was also studied as a 
function of deuteron dose. A sample of the findings for the degeneration of 
the spike potential due to different doses of deuterons is presented in Fig. 6. 
Deuteron experiments, which were carried out in the spring and summer 
seasons, showed only a small enhancement of neural output. 




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■■■IIK^I 

■■i'i 



■■■■■■ ■■■^■■■■■i 

ili|ii ■■■«!■■■■■■ 

■■■■■■ ■■■■■■■■■■ 




■■nil 
sami 

■■II 

2 hr 



B^^riiKiiii 
■■i»llli 
■■■■»^i 





10 hr 12 hr 14 hr 

Fig. 2. A composition of action potentials from the right and left sciatic nerves 
of a frog before and after irradiation. The nerve producing the action potentials in 
the upper section of each row was exposed to 72 krad of 910 Mev alpha particles. The 
nerve producing the action potentials in the lower section of each row served as a 
control. Conduction block occurred in the bombarded nerve 14 hours following irradi- 
ation. The oscillograms have 8 units on the vertical axis and 10 units on the hori- 
zontal axis. On the ordinate, 1 unit is equivalent to 2.5 mv; on the abscissa, 1 unit is 
equivalent to 1 msec. 

281 



282 



C. T. GAFFEY 




2 4 6 8 10 12 14 16 

Hours after alpha particle irradiation 

Fig. 3. The percent of the initial spike potential is plotted against postirradiation 
time for a nerve exposed to 72 krad of alpha particles and for its pair control. The 
oscillograms in Fig. 2 provided the data for the construction of Fig. 3. 



140 

_ 120 
g 

Sioo- 

o 

Q. 

« 80 
'5. 

r 60 



Pre-irrodiotion 



20- 339 krad 




2 4 6 8 10 12 

Hours after alpha particle irradiation 

Fig. 4. Percent of initial spike potential plotted against the time after irradiation 
by 910 Mev alpha particles for doses between 2 and 339 krad. 



The relative inhibitory effects of alpha particle and deuteron irradiation 
on excitability are exemplified in Fig. 7. The time for the complete extinction 
of spike amplitude is a logarithmic function of the absorbed dose within cer- 
tain limits. Below 30 krad for alpha particles and 60 krad for deuterons, no 
demonstrable suppression of the spike potential of sciatic nerve due to irradi- 
ation can be reported. Irradiated nerves after more than 24 hours showed 



HIGH ENERGY IRRADIATION: BIOELECTRIC EFFECTS 



283 




2 4 6 8 10 

Hours for non-irradiated nerves 



Fig. 5. Nonirradiated control nerves exhibit a variation in the amplitude of the 
spike potential when plotted as percentage of the initial spike potential. 




024 680 12 14 

Hours after deuteron irradiation 

Fig. 6. The time course for the inhibition of the spike potential of nerves bom- 
barded with 455 Mev deuterons in the dose range 50 to 300 krad is pictured. Altera- 
tions in the magnitude of the action potentials are given in terms of relative spike 
activity, i.e., percentage of the initial spike potential. 

deterioration of spike potential activity, but the degree of impairment was 
mimicked by the nonirradiated controls. The slope of the alpha particle dose- 
survival line is double that of the deuteron line (Fig. 7). This alpha particle- 
deuteron slope ratio is taken as evidence that alpha particles have twice the 
relative biologic efTectiveness of deuterons. 

Conduction velocities of propagated impulses have been computed from 



284 



C. T. GAFFEY 




4 8 12 16 20 24 28 

Hours after irradiation for action potential toss 

Fig. 7. The logarithm of the dose of irradiation (910 Mev alpha particles and 455 
Mev deuterons) is plotted against the survival time of neural excitability. High energy 
particles given in doses near 300 krad promptly inhibit the action potential of frog 
sciatic nerve. Above 100 krad each point on the diagram is the mean of 2 experiments; 
below 100 krad each point is the average of 3 experiments. 



the time delay between 2 spike peaks on oscillograms and the distance be- 
tween recording electrodes. Alterations from irradiation in conduction veloc- 
ity, latent period, and stimulus strength do not appear strongly related to 
suppression of the spike amplitude, because when the propagated impulse 
was 90% abolished, conduction velocity was retarded by only 25 to 30% of 
its original value, and the stimulus strength and latency period were changed 
by approximately 25 and 20%. From recent studies employing 2 Grass stimu- 
lators (Model S-4), it was found that the refractory period increases (after 
a small transient decrease) before conduction velocity reduction, action po- 
tential depression, latency period prolongation, and stimulus strength altera- 
tions and is, thus, the earliest index of radiation damage that the author 
has noted. 



Dose Rate Studies 

The influence of modifying the dose rate at which alpha particles were 
administered to isolated sciatic nerve was investigated. The cyclotron's beam 
was adjusted to deliver high energy particles at the rate of 0.5, 1.0, 2.0, 4.0, 
and 8.0 krad per minute in 8 experiments. The survival of excitability was 
found to be independent of the intensity at which irradiation was absorbed 



HIGH ENERGY IRRADIATION: BIOELECTRIC EFFECTS 



285 



and dependent on the quantity of dose absorbed. The inhibition of neural 
activity resulting from irradiation was not reversible. 

Radioactive Studies 

The influence of alpha particle irradiation on sodium ion permeability of 
sciatic nerve can be presented here only as a brief, preliminary report. Nerve 
sheaths were left intact in order to prevent volume changes. 

Long life Na^^ was used as a radioactive tracer in Ringer's solution ( 1 )u.c 
per ml of Na-^). The proximal ends of isolated nerves were ligated with 3 
mil tantalum wire to allow manipulation of the nerves. To determine the 
time course for the penetration of radioactive sodium, the nerve was im- 
mersed in "hot" Ringer's solution, and the activity accumulated during this 
soaking period was estimated by removing the nerve from the Na-^ Ringer's 
solution to a 4 ml vial of nonlabeled Ringer's. A scintillation spectrometer 
registered the radioactivity of the sample, and the nerve was restored to the 
Na^" Ringer's solution for additional radioactive tracer uptake. The Na^^ 
which diffused from the nerve during the counting time could subsequently 
be estimated by recounting the vial of contaminated Ringer's solution. 

Results revealed that nerves given less than 150 krad of alpha particle irra- 
diation did not differ significantly from their nonirradiated controls in the 
kinetics of sodium penetration. In the dose range 150-200 krad, the rate of 
Na-^ uptake for irradiated ner\es was increased to only a small extent over 
controls (Fig. 8) . 



7x10^ 




80 

Minutes 



160 



Fig. 8. The time course for the entry of radioactive tracer sodium into sciatic 
nerve exposed to a 150 krad dose of 910 Mev alpha particles and its pair control. 
The experimental temperature was 21°C±1.5°C. 



286 



C. T. GAFFEY 



The technique for studying the emergence of Na^^ from isolated sciatic 
nerve was similar to that described by Shanes (1954). Nerves were immersed 
in Na^- Ringer's solution for approximately 12 hours at 10° C, brought to 
room temperature (21° C), and irradiated in the beam of the 184-in. syn- 
chrocyclotron. The emergence of Na'^ from the "loaded" nerves into fre- 
quently replaced vials of inactive Ringer's was measured with a satisfactory 
degree of accuracy (counting error less than 1%) by a scintillation spec- 
trometer. 

Figure 9 illustrates that after an exposure to 200 krad of alpha particle 
irradiation, there was a small decrease in the rate of movement of sodium 
ions from the irradiated nerve when compared to its control. From 8 experi- 
ments in which nerves were administered doses below 150 krad of alpha 
particles, there was no evidence of an alteration in the rates of loss of Na^^ 
as a consequence of irradiation. 

From these limited radioactive studies, it can be inferred that with alpha 
particle irradiation in excess of 150 krad there is probably a rise in the so- 
dium ion content of sciatic nerve due to an increase in the rate of sodium ion 
penetration coupled with a decrease in the rate of sodium ion loss. 

In these experiments the studies on the rate of Na^' loss began 5 minutes 
after irradiation was completed, while Na^- uptake studies started 1 hour 
pjostirradiation. 



100 



.2 10 




40 



80 120 

Minutes 



160 



200 



Fig. 9. Decline of the Na" content (percent initial) of sheathed sciatic nerves by 
diffusion into Ringer's solution. Each of the experimental points on the 200 krad 
alpha particle line is the average data from 4 experiments, as are the points on the 
control line. 



HIGH ENERGY IRRADIATION: BIOELECTRIC EFFECTS 287 

Discussion 

The present findings indicate that irradiation of frog nerve with 30 krad 
or less of high energy alpha particles or deuterons was below the minimal 
dose required to evoke an early impaimient of neural activity. Such a result 
is in general agreement with other obsenations found in the literature. 
Schmitz and Schaefer (1933) reported no functional damage to frog sciatic 
ner\e when exposed to 10 kr or x-rays. For rat sciatic nerve, no apparent 
effect on neural conduction after exposure to 10 kr of x-rays has been ob- 
served (Janzen and Warren, 1942). Similarly, Rothenberg (1950) adminis- 
tered 50 kr to squid's stellar axon and reported that when the preparation 
was electrically stimulated, good action potentials were present. From the 
data offered in this paper, it is reasonable to report that 30 krad of 910 Mev 
alpha particles represents a threshold dose for the destruction of bioelectrical 
activity of the amphibian nerve. An explanation on the molecular level 
which w'ould account for what determines the functional resistivity of nerve 
to ionizing radiation is not found in the literature. 

For a prompt inhibition of bioelectric activity of frog sciatic nerve, about 
300 krad of alpha particles or deuterons was required. Using a frog muscle- 
nerve preparation, Audiat (1932) and Audiat et al., (1934) observed that 
administering 300 kr of x-rays caused a loss of neural excitability. Gerstner 
(1955; Gerstner et al., 1956) stated that the sciatic nerve of bullfrog {Rana 
catesheiana) suffered a conduction block when exposed to about 300 kr of 
high intensity x-radiation. The neural alterations of mammalian nerve 
during x-irradiation have been investigated by Bachofer (1957) and Bachofer 
and Gartereaux (1960a, b), and they established that approximately 500 kr 
will extinguish the amplitude of the spike potential of the ventral caudal 
nerve of the rat. Extirpation of axonal activity of the median and lateral 
single giant nerve fibers of the earthwomi {Lumhricus terrestris) was shown 
to occur after 246 and 306 kr of x-rays (Bachofer and Gautereaux, 1959). 
The neural mechanisms affected by these massive doses of irradiation have 
not been established. 

The sodium influx into squid giant axon immediately after x-irradiation 
has been reported by Rothenberg (1950) using Na-*. After 125 kr, sodium 
influx was increased markedly. On exposure to 50 kr, the rise in sodium 
permeability was smaller, but significant. The Na^^ experiments on frog 
nerves (described in this report) after alpha particle irradiation are in 
harmony with the view that irradiation increases sodium ion permeability. 
However, the alpha particle dose must be near 150 krad to express a sodium 
permeability increase. 

Experiments have revealed that the relative biologic effectiveness (RBE) 
of alpha particles is twice that of deuterons in inhibiting neural activity. It 



288 C. T. GAFFEY 

is known (Zirkle, 1954) that the linear energy transfer (i.e., the stopping 
power or rate of energy loss) along a particle's track varies as the square of 
its charge. The linear energy transfer on an alpha particle is 4 times that of 
a deuteron of the same velocity. Since biologic eflfects in general vary with 
the linear energy transfer, it would be expected that the RBE of alpha 
particles with respect to deuterons would approach 4 as a limit. 

Membrane Model 

In the following section a membrane model for nerve is outlined with the 
hope that such a model may suggest how the function of nerve is affected by 
radiation energy. 

Direct evidence of neural membrane structure, in terms of lipid and 
protein components, must await a detailed study of lipids and lipid-protein 
systems. Whatever may be the ultimate interpretation of the molecular or- 
ganization of the axon membrane, it is probably safe to say from electron 
microscope studies that the unit membrane includes two protein monolayers 
allied with a double layer of lipid molecules (Schmitt, 1959) . 

If it is assumed that the protein molecules of the neural membrane are 
helical in nature and form an oriented structural layer, certain insights into 
membrane properties are revealed. When 3 protein molecules of macro- 
molecular diameter are closely packed, a 4th element is created — an inter- 
stice or fault which for convenience will be referred to as a "channel." When 
3 protein macromolecules 28.2 A in radius are most efficiently packed, an 
intermolecular channel about 4 A in radius is obtained (Fig. 10). 

It is known that models of membranes based on the concept of a continu- 
ous lipid layer are untenable because experiments reveal that biologic mem- 
branes are crossed by molecules of water and numerous compounds insoluble 
in fat. This is a property of a membrane with channels rather than a solution 
process in a lipid film. Comparison of rates of water entrance into cells 
under the influence of osmotic pressure gradients and simple difTusion 
gradients gives a rough indication of what may be the "equivalent channel 
size" (KoeflFed- Johnson and Ussing, 1953; Prescott and Zeuthen, 1953). 
Values of channels range from 5 A in red blood cells to 16 A in squid axons 
(Nevis, 1957; Solomon et al., 1957) . When frog nerve is placed in a medium 
labeled with deuterium, tritium or O^^, the half time for equilibration is 
only 1 minute (Tobias and Nelson, 1959). Hence, the existence of a channel 
pathway through the ultrastructure of cell membranes to water and small 
ions seems likely. 

It has been suggested by Mullins (1956) that the number of water mole- 
cules associated with each ion in traversing the neural membraine is limited 
to the same minimum, say one. In physiologic solutions sodium ions are 



HIGH ENERGY IRRADIATION: BIOELECTRIC EFFECTS 289 




K* 
Macromolecule 



Fig. 10. Protein macromolecules are schematically represented as circles oriented 
hexagonally. At the junction of 3 macromolecules, an interstice is formed which in 
the three-dimensional model would be a channel. Drawn to scale it can be seen that 
potassium with its primary layer of hydration fits the channel created by the 
macromolecules. 



considered to be larger than potassium ions, because sodium ions orient more 
layers of hydration due to the intense electric field created by the charge on 
the ion (Ling, 1952, 1957). Potassium ions with a lower energy of hydration 
than sodium have effectively fewer oriented shells of hydration and hence a 
higher mobility in an aqueous medium. Kortum and Bochris (1951) p>oint 
out that for cations, water molecules on the first layer (primary hydration) 
are held so tightly that the primary hydration shell moves as a unit with the 
ion. However, water molecules beyond the primary hydration layer are 
loosely oriented and exchange readily with surrounding water molecules. 
Hence, it is an acceptable hypothesis that ions with one layer of hydration 
migrate through neural membranes. From Fig. 11 it is seen that the radius 
of primary hydrated potassium is 4.05 A which is larger than primary hy- 
drated sodium (3.67 A in radius; Fig. 12) . The crystal lattice radii are taken 
from Pauling (1945), and the width of concentric water shells of hydration 
is that of the diameter of a water molecule, 2.72 A (Buswell and Rodebush, 
1956). 

The intermolecular forces between protein elements of the membrane are 
no doubt subjected to lateral straining pressures produced by thermal motion 
(kinetic and vibrational) and cytoplasmic streaming. As a consequence, a 
channel is never a fixed size, but statistically distributed, probably in a 
Gaussian fashion. The mode of the channel size distribution of the resting 
neural membrane is assigned to the ion empirically known to have the high- 



290 



C. T. GAFFEY 




Radii Hydration energy 
/j^ \ (^) (kcal/Mole) 

...4.05 34.5 

-2 6.77 16.3 

-3- 9.49 6.9 

J -...12.21 3.9 

5-^00 ...00 13.9 

Fig. 11. Representation of the potassium ion with a crystalline radius of 1.33 A 
and the 1st, 2nd, 3rd, and 4th hydration shells. The diameter of a water layer is 
taken as 2.72 A. Hydration energies for a given hydration shell are computed on the 
basis that hydration energy exponentially decreases with the distance from the charge 
on the ion. 




Hydration energy 
^r-x \ Radii (A) (kcol/Mole ) 
(I^-... J.... 0.95 

X-^. 3.67 49.8 

_2._ 6.39 19.0 

-3.-- 9.11 77 

J*.- 11.83 4.1 

5-*-oo 00 13.9 

Fig. 12. Representation of the sodium ion with a crystalline radius of 0.95 A and 
the 1st, 2nd, 3rd, and 4th hydration shells. 



est relative membrane permeability, potassium (see Fig. 13, resting state). 
The spread of the Gaussian distribution curve for the resting state is adjusted 
so the area representing potassium ion channel is 25 times greater than the 
area representing sodium ion channels, which is in harmony with Hodgkin 
and Katz's (1949) evidence that the relative permeability of potassium to 



HIGH ENERGY IRRADIATION: BIOELECTRIC EFFECTS 



291 




3 2 3.4 3.6 3.8 4D 4.2 4.4 4.6 

Ionic radius ( A) 

Fig. 13. The relative permeability of an ion (ordinate) is considered a physiological 
term interchangeable with density of channel size. The ordinate on these diagrams 
could just as conveniently read "number of available channels per unit area of mem- 
brane." It is a reasonable assumption that channel size (abscissa) is distributed ac- 
cording to a Gaussian curve. For neural membranes it is assumed the mode of the 
distribution curve is (a) in the resting state that of a potassium ion, (b) in the con- 
ducting state that of a sodium ion, and (c) in the irradiation state somewhere between 
(a) and (b). 



sodium ions across the axonal membrane is 25 to 1. During excitation these 
relations are reversed, i.e., the permeability of potassium to sodium is 1 to 
25. Hence, the mode of the distribution curve during activity is assigned to 
sodium (Fig. 13, conduction state) . 

The concept that the neural membrane behaves as though it were a 
molecular sieve is not practical. Such a proposal does not offer an explana- 
tion of how the cell discriminates between potassium and sodium ions as 
indicated by p>ermeability studies. A molecular sieve model for the membrane 
permits a small ion to pass through any channel of greater size than itself. 
On this basis, sodium ions 3.67 A in radius should have free permit through 
channels that sterically just pass potassium ions 4.05 A in radius. Hence, a 
molecular sieve model fails to explain selective ion permeability. This diffi- 
culty is removed if solvation (interaction with membrane components) is 
"quantized." That is, an ion on entering a channel has all of its hydration 
shells beyond the primary hydration level solvated by the wall of the 



292 C. T. GAFFEY 

channel (Mullins, 1956). It is maintained that only whole shells of hydration 
for ions are replacable by the membrane's wall in the process of solvation. 
In this sense, a 3.67 A sodium ion would not "fit" into a 4.05 A potassium 
ion channel because the quantum solvation provided by the wall of the 
potassium ion channel does not match a sodium ion. This could be true only 
if some level of hydration (secondary, tertiary) for sodium did uniquely 
match some hydrated size of potassium. By similar reasoning, potassium 
ions do not fit sodium size channels. A comparison to Bohr's theory in which 
electrons exist in integral energy levels is only an analogy, but it may help 
one's thinking. For the membrane's channel wall, it is held that instead of 
an infinite number of solvation levels, there are only a restricted number 
with properties represented by functions of n, where n is an integer. 

If the quantized view of membrane solvation is correct, and ions penetrate 
the membrane with only the primary layer of hydration, then it will cost the 
cell more in solvation energy to transport sodium (44.7 kcal/mole) than 
potassium (40.5 kcal/mole) . On the evolutionary scale it would appear that 
the cheaper ion was selected to balance the intracellular negative charges. 

The initial suggestion of a helical protein structure does not violate our 
knowledge about the architecture of proteins. In terms of our membrane 
model, the helical nature of protein provides a key to the interpretation of 
the excitation phenomena of axons. Protein membrane molecules are con- 
ceived to be in a contracted or coiled state, while the nerve is in the resting 
state. A threshold stimulus permits the constrained, helical macromolecule 
to become relaxed or uncocked, thus diminishing the radius of the macro- 
molecule. Intermolecular attractive forces maintaining membrane structural 
order cause a decrease in the mode of the channel size distribution when 
coiled macromolecules uncock. If, on stimulation, the membrane macromole- 
cules alter their radii from 28.2 A to 26.2 A, the new mode of the channel 
size distribution will be 3.67 A, the size of primary hydrated sodium ion (see 
Fig. 13, conducting state). It is naive to imagine that the helical protein 
molecules have characteristics of a mechanical spring. A coiled spring can be 
stretched a good deal before a decrease in radius is effected. The reduction 
of the radical dimension of the membrane's helical molecules is perhaps due 
to the action of London forces. 

A pleasing consequence of this membrane model is the number of neural 
characteristics it can interpret. The all-or-none law for axons on the 
molecular level can be viewed as the states which the membrane helical 
macromolecules can occupy; either a stimulus is sufficient to uncock the 
macromolecule, or it is not. If the stimulus is sufficient, the channel size 
mode shifts from potassium to sodium and ions follow their electrochemical 
gradient generating a bioelectric impulse. Molecularly translated, the refrac- 



HIGH ENERGY IRRADIATION: BIOELECTRIC EFFECTS 293 

tory p)eriod of a nerve is the time element required to restore the helical 
macromolecule to the constrained state. 

Hodgkin and Katz (1949) have presented evidence show^ing that at rest 
the ionic permeability of potassium is 25 times that of sodium. During 
excitation these ionic permeabilities are quickly reversed, so that sodium is 
25 times as permeable as potassium. The burden of accounting for this 
sudden ionic shift has undone many an ingenious membrane hyp>othesis. The 
cocked-uncocked f>erformance of helical molecules in the present membrane 
model supplies an adequate explanation not only for the permeability events 
triggered by excitation, but also for the time constants for the limbs of the 
action potential. 

The character of the cocked protein molecule is such as to allow for a 
rapid change to the relaxed structural state, thus accounting for the fast 
time constant of the ascending limb of the action potential. To "reconstrain" 
the relaxed helical macromolecules suggests a need for an energy input. 
Since the ratio of heat produced during activity over that produced in re- 
covery shows that the latter requires most of the energy, we have another 
observation that does not violate the model, but agrees with it. 

Developing a membrane model has made the task of interpreting how 
radiation energy influences neural functioning, relatively easy. Only an 
average energy of some tens of electron volts can be accepted by a molecule. 
Successive energy transfers occurring along the path of high energy particles 
(kinetic energies in the thousand or million electron volt range) supersede 
the acceptable energy level, and a defective molecule is the consequence. For 
conducting axons, it is construed that the most radiation-labile molecules are 
the membrane protein macromolecules. When the structure of these mole- 
cules is damaged, excitability is affected. 

Radioactive tracer studies of resting nerve (Rothenburg, 1950; also see 
Radioactive Studies in this paper) reveal that radiation causes an increase 
in sodium ion permeability, i.e., a shift in the mode channel size from potas- 
sium toward sodium (see Fig. 13, resting state versus irradiation state). 
Bioelectric studies on single myelinated nerve fibers after irradiation (Gaffey, 
1960) indicate that there is an increase in potassium ion permeability 
(revealed by a decrease in the slope of the falling limb of the action poten- 
tial) and a decrease in sodium ion pemieability (connoted by a decrease in 
the slope of the rising limb of the action potential). The deterioration of 
selective ion permeability in the conducting nerve is viewed as a translation 
in the mode channel size from sodium toward potassium (Fig. 13, conduct- 
ing state versus irradiation state). These early signs of neural impairment 
would be expected as a consequence of the partial loss of the ability of the 
helical molecules of the membrane to fully coil-uncoil. The terminal degener- 



294 C. T. GAFFEY 

ative steps due to radiation take place rapidly and could be interpreted as a 
full loss of the membranes' helical molecules capacity to change states, per- 
haps as a result of a loosening of their structure. This would cause a broad- 
ening and flattening of the channel size distribution curve, which in essence 
eliminates selective ion permeability, thus producing a rapid loss of excitability. 
In conclusion, it can be argued that certain doses of radiation are thresh- 
old for neural injury as a result of the membrane's macromolecules being 
irreversibly impaired in their ability to change states. 

Summary 

Isolated sciatic neives from Rana pipiens were exposed to cyclotron ac- 
celerated beams of 455 Mev deuterons and 910 Mev alpha particles. The 
degree of electro-physiologic damage was found to depend on the dose of 
irradiation and the elapsed time from irradiation. 

With massive doses of alpha particle or deuterons (greater than 300 krad) 
the action potential of the frog's sciatic nerve was promptly suppressed. 

Within the range 30 to 300 krad for alpha particles and 60 to 300 krad for 
deuterons, the survival time of the action potential was a logarithmic func- 
tion of the absorbed dose. 

Alpha particles were found to have twice the relative biologic efTectiveness 
of deuterons in blocking excitation. 

An increase in the refractory period was manifested before conduction 
velocity reduction, action potential depression, latency period prolongation, 
and stimulus strength alteration in the high energy irradiated nerve. 

Alpha particle irradiated nerves were shown not to increase in sodium ion 
permeability for doses less than 150 krad. Between 150-200 krad the rate of 
Na^^ penetration was slightly increased, while the rate of loss of Na-- from 
the nerve was decreased. 

Variations in the exposure rate from 0.5 to 8.0 krad per min failed to 
induce a dose-rate effect for alpha particles. 

The inhibition of neural activity resulting from alpha particle and deu- 
teron irradiation was not reversible. 

A model for the neural membrane was outlined, and the action of radia- 
tion was interpreted on the basis of this model. 



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HIGH ENERGY IRRADIATION: BIOELECTRIC EFFECTS 295 

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Morphologic and Pathophysiologic Signs 

of the Response of the Nervous System 

to Ionizing Radiation 

(Review of main works published in the USSR) 

N. I. Grashchenkov* 

Institute of the Higher Nervous Activity, 
U.S.S.R. Academy of Sciences, Moscow, U.S.S.R. 

Introduction 

The decisive role in the complex interrelationship between animal organ- 
isms (including human) and their environment belongs to the nervous 
system. All environmental agents, whatever their nature, address themselves 
in the first instance to the nervous system. 

The degree to which an animal or human organism reacts to various 
harmful stimuli is determined by the properties of functional mobility and 
working unity of the vegetative nervous system with the endocrine system of 
the organism and by the interdependence between these systems and the 
peripheral and central nervous system. 

During the last few decades a new environmental factor has appeared — 
ionizing radiation. The degree of concentration of this radiation in the en- 
vironment is inevitably increasing owing to the further development of 
atomic weapons, the growth of powerful installations, electric power plants, 
ships, and submarines which process radioactive substances, and the increas- 
ing quantity of scientific research using radioactive substances. The result is 
that an ever increasing number of people are being subjected to the action of 
ionizing radiation. 

To protect man from the harmful action of ionizing radiation we need 
thorough investigation and accurate knowledge of the mechanism by which 
ionizing radiation acts on the animal and human organisms. Once this is 
known, a series of preventive and therapeutic means can be suggested which 
would eliminate or reduce the force of the harmful action of ionizing radia- 
tion on the organism as a whole or on particular systems and organs. It is 
generally admitted that ionizing radiation attacks the human organism first 

* Much of the work entailed in compiHng this review and the attached bibhography 
has been done by the staff of the laboratory of Dr. B. M. Hecht and Dr. A. M. Weln. 

297 



298 N. I. GRASHCHENKOV 

and chiefly through the nervous system, in particular the vegetative nervous 
system, and the blood and its principal morphologic elements. 

Owing to the special conditions under which physiology has been devel- 
oping in the U.S.S.R. and the exceptional attention paid to the leading role 
of the central nervous system in the physiologic functions and in the de- 
velopment of disease pathogenesis in man, a great deal of research in the 
U.S.S.R. has been devoted to the morphologic and structural changes which 
represent the nervous system's response to ionizing radiation. 

This review does not cover all the published literature but merely gives 
an idea of some of the main publications on this subject. 

Morphologic Changes in the Nervous System 
Under the Effect of Ionizing Radiation 

Our ideas about the resistance of the nervous system to radiation are based 
mainly on the negative morphologic picture observed after irradiation. A 
definite discrepancy between the morphologic and the functional shifts has 
come to light, providing evidence as to how the nervous system reacts to 
radiation. A number of factors lie at the root of these apparent contradic- 
tions. For one thing the methods in use today for studying the morphology 
of the nervous system are fairly crude and not always adequate for the pur- 
pose of estimating slight shifts occurring in the tissues. Progress in this field, 
therefore, depends on the use of more precise methods of investigation, 
particularly histochemical methods. Further, in cases of extremely serious 
lesions, the death of the animal occurs quickly, and the morphologic changes 
do not have a chance to develop over a long period. Finally, in recent years 
fairly definite morphologic changes which account for shifts of a neuro- 
dynamic character have been demonstrated. 

In the opinion of many Soviet investigators, the term radiosensitivity is 
not a happy expression. It would be more correct to speak of radiore activity, 
which would be described in terms of a change in some functional reaction 
on the part of the nervous system, and of susceptibility to radiation damage, 
which would be described in terms of morphologic symptoms. 

When animals are subjected to large doses of radiation which produce 
the pattern of radiation disease, the following phenomena are seen in the 
central nervous system: swelling of the cells, argentophilia, displacement of 
the chromophil substance at the height of the disease, and hemorrhagic 
diathesis, often in the thickness of the meninges. Degenerative necrotic 
changes, such as vacuolation of the ganglionic cells, increased precipitation 
of silver, distension of the cells, and changes in the processes (Kraevsky, 
1957), are observed primarily in the zones of the higher vegetative subcorti- 
cal centers. Some authors have not detected any reaction on the part of the 
microglia (Kraevsky, 1957), while others found dystrophic changes (Alek- 
sandrovskaya, 1959). 



MORPHOLOGIC AND PATHOPHYSIOLOGIC SIGNS 299 

It has been demonstrated that there is a relationship between the size of 
the radiation dose and the character of the morphologic changes (Aleksan- 
drovskaya, 1959) . With a dose of 650 r, cerebral anemia occurs macroscop- 
ically, with hyperemia in some parts of the brain. Histologically, dystrophic 
changes can be detected in the cortex, subcortical ganglia, and hypothala- 
mus and vacuolation accompanies partial destruction of the nerve elements 
in the corpora quadrigemina, nuclei of cranial nerves, and structures of the 
reticular formation. Swelling or edema becomes established, and the nerve 
fibers break up. Wrinkling of the astrocytes takes place. Aleksandrovskaya 
(1959) characterizes the above picture as toxicohypoxic encephalopathy 
with dystrophic changes of the nerve cells and productively dystrophic re- 
actions on the part of the glia. 

All these changes were deeper and more diffuse after irradiation with a 
dose of 1,300 r. Subarachnoid and perivascular hemorrhages were observed. 
Portugalov (1957) found the most substantial changes in the diencephalon 
and mesencephalon. Several authors have pointed to morphologic changes 
(necrobiosis) of particular cell groups in the cortex, accompanied by chro- 
matolytic and karyolytic phenomena [Kurkovsky, 1958 (see in this review 
the following authors : Kyandaryan ; Papoyan ; Beglai7an, Zagotskaya, Aruty- 
anyan, 1957)]. Bibikova (1959) obtained acute or chronic demyelination 
after irradiating dogs with 5,000-30,000 r. 

Smirnov (1960) observed changes in the distribution and quantity of 
tigroid substance (Nissl bodies) in the neurons of the reflex arc, an index to 
the functional condition of a neuron. 

Manina (1959) demonstrated the destruction of neuroblasts and severe 
disturbance of the architectonics in different parts of the cell as a result of 
irradiation with radioactive phosphorus (P^-). 

Definite morphologic shifts occur under the effect not only of large doses 
of radiation but also of single or repeated small doses (Aleksandrovskaya, 
1957) . These shifts take the form of proliferation responses by microglia and 
ectodermal glia. Profound morphologic changes are observed in the progeny 
of rats irradiated with a dose of 150-200 r during the antenatal jDcriod. 

Shabadash (1957) has demonstrated that irradiation with a dose of 
25-100 r causes a shift of — 0.3 to — 0.4 in the pH of the nucleoproteins. 
The response is detected first in the mitochondria, then in the tigroid sub- 
stance. These changes are caused by depolymerization processes and reduc- 
tion in the quantity of nitrous bases. A typical feature is that the huge 
number of mitochondria in the diencephalic region and the reticidar fornia- 
tion suflfer from ribonucleoprotein deficiency as a result of irradiation; this 
indicates local respiratory disorders. There is undoubtedly a good prospect 
that this cytochemical change will be useful in revealing the mechanism by 
which ionizing radiation afTects the organism. 

These findings are evidence that morphologic changes occur in the brain 



300 N. I. GRASHCHENKOV 

after radiation. Not all sections, however, are equally damaged (the marked 
changes in the higher vegetative centers of the hypothalamus are note- 
worthy) , nor are all the structures which form part of a given section equally 
involved. Portugalov (1957) attempted to account for this on the grounds 
that those microstructures which were in an active condition at the moment 
of irradiation suffered most damage. 

It has also been demonstrated that there are two stages in the development 
of morphologic changes in the brain which occur during the first days of the 
disease and at its climax. In the first period, the changes in the nervous 
system are regarded as the result of direct action by radiation on the nerve 
elements. As to the second period (late necroses), some authors are inclined 
to regard these changes as the result of a hemodynamic disorder, while others 
consider them the result of a metabolic disorder and a manifestation of 
toxic elements. The latter hypothesis seems to be better founded, since 
sometimes there are no hemodynamic disorders, while it has been demon- 
strated that definite substances of lipoid origin, hemolysins, are present. They 
are formed when tissues are subject to direct irradiation (Tarusov, 1957; 
Benevolensky, 1 95 7 ) . 

The radiosensitivity of nerve elements in young animals and embryos is 
not disputed, and there is a considerable amount of literature to attest to it 
(Zavarzin et al., 1936; Emdin and Shefer, 1935; Olenev and Pushnitsyna, 
1952; Manina, 1959; Aleksandrovskaya, 1959). 

It is possible to determine the morphologic changes which occur in the 
peripheral nervous system during irradiation of the organism. Oleinikova 
(1936) observed degenerative changes in the nerve trunks only 30-60 min- 
utes after irradiation. These changes reached a peak at the height of the 
radiation disease. The nerve fibers broke up into segments; some were in a 
state of granular decay, and partial demyelination phenomena were observed. 
Changes were also noted in the cells of the Auerbach and Meissner plexuses. 
In 1953, Lebedev showed that Auerbach's plexus was involved and that 
Meissner's plexus had great stability. He noted, however, reactive and de- 
structive changes in the sensory cerebrospinal and craniocerebral (trigeminal 
and vagal) ganglia and in the sympathetic ganglia. 

Anisimova and Aleksandrova (1959) consider the afferent nerve channels 
and their terminal receptor apparatus to be the most sensitive. Kraevsky 
(1957) also notes that of all the parts of the peripheral nervous system it 
is the peripheral parts of the sympathetic nervous system which are most 
susceptible. Gracheva (1959) is the only author who has not detected con- 
vincing morphologic changes in the nerve cells of the vegetative and sensory 
ganglia. Damage to the peripheral nervous system, accordingly, consists 
primarily in disorders of the constituent parts of the vegetative nervous 
system. 



MORPHOLOGIC AND PATHOPHYSIOLOGIC SIGNS 301 

Pathologic Signs of the Action of Ionizing Radiation 

One cannot discuss the effect of ionizing radiation on the functioning of 
the nervous system without mentioning the early research on this subject 
in the Pavlov-Nemenov laboratories in the late 1920's. This was the period 
when the view that the nervous system is highly radioresistant was widely 
propagated in the literature — an opinion held to this day by a great many 
foreign authors, but based on the morphologic research of that time. 

Nemenov (1932, 1950), however, by using the conditioned reflex method 
was able to demonstrate that this view was incorrect and to provide con- 
firmation that the nervous system is radiosensitive. Today we have accumu- 
lated extensive information on disturbances of the higher nervous activity by 
exposing various experimental animals and human subjects to radiation and 
studying its effect on their conditioned reflex activity. As this material has 
been dealt with in a number of reviews and monographs, I shall confine 
myself to outlining the results obtained from work done in the last few years. 

These researches have shown that various types of ionizing radiation lead 
to phase changes in the neurodynamics of the cortex. During the initial 
periods the cortical activity is stimulated, but later, depression sets in. The 
duration of each period depends both on the condition of the nervous system 
and on the dose and characteristics of the radiation. 

In recent years the work of Kotlyarevsky and co-workers (1957), Livshits 
(1955, 1956a, b), Lomonos (1953), and Piontkovsky and co-workers (1957) 
has provided confirmation that phase changes occur in the higher nervous 
activity after whole body exposure of experimental animals to small or 
medium doses of radiation. Lomonos (1959) noted phase changes in the 
conditioned reflex activity after the body of a dog had been exposed first to 
a single and then to repeated doses of radiation with its head shielded. In 
several cases, prolonged disturbances of the conditioned reflex activity have 
been observed without any clinical signs of radiation sickness. 

Klimova (1957) observed persistent changes in the higher nervous activity 
without clinical symptoms of radiation sickness following internal irradiation 
of animals (dogs) fed 1 ^c each of radioactive strontium for 6 months. She 
found that the conditioned reflex activity was at first heightened, then 
depressed, and that phase conditions appeared in the cerebral cortex. Four 
to 6 months after irradiation, similar disturbances were observed in the 
unconditioned reflexes. Restoration of the nervous activity took place un- 
evenly. The motor defense reflex was the first to return to normal at the end 
of the 1st month, followed by the motor food reflex, and 2-2/2 months later 
by conditioned reflex activity. Various defects were still observed in the 
dogs' higher nervous activity even 6 months after the exj>eriment. 

With large doses of ionizing radiation, the initial phase of cortical activa- 



302 N. I. GRASHCHENKOV 

tion may not occur. Minaev (1957), for example, observed no phase during 
which the excitability of the brain increased when he exposed the head of a 
dog to a dose of 7,000 r. 

Of great practical interest is the research which has been done on the 
influence of small doses of ionizing radiation on the conditioned reflex 
activity of the brain. More and more evidence is accumulating to indicate 
that small doses of 5 to 10 to 20 r lead to changes in the higher nervous 
activity, mainly to disturbance of internal inhibition, disinhibition of the 
capacity to differentiate, and disturbance of the inhibiting conditioned 
reflexes. Gorsheleva (1958), Airapetyants (1958), Malyukova (1958), and 
Rokotova and Gorbunova (1958) noted a change in the conditioned reflex 
activity after the administration of an even smaller dose (2 r). 

Even such small doses of radiation are capable of having a cumulative 
effect. Malyukova (1958) and Meizerov (1958) have shown that systematic 
exposure to x-rays in doses of 3-15 r led to serious disturbances of the higher 
nervous activity when the total dose reached 130-190 r. Tests performed on 
human subjects by working with radiation doses approaching the permissible 
limit and using the speech-motor method have also revealed definite disturb- 
ances of the higher nervous activity : delay in forming the conditioned reflex 
connections, prolongation of the latent period of the conditioned reflex, and 
a number of other signs indicating loss of mobility and equilibrium in the 
nervous processes (Morozov, Drogichina et al., 1957). 

Voevodina (as quoted by Cherkasov, 1960) has shown that even after 
rats have been exposed to x-radiation in a 10 r dose, the conditioned reflex 
activity is disturbed. After the rats had been exposed to a triple dose of 10 r, 
the conditioned reflexes could not be elicited for 42 days. Prolongation of the 
latent period and disturbance of the capacity to differentiate were still ob- 
served for the next 6 months. Even many months after the brain had been 
exposed to this dose, functional weakening of the nervous system caused by 
the injection of morphine again led to disturbance of the conditioned reflex 
activity. 

Work done in Kupalov's laboratory established the fact that after animals 
were subjected to small doses of radiation, aminazine (equivalent to chlor- 
promazine) in a dose of 0.5 mg per kg caused acute excitation in the animal, 
with serious disturbance of the conditioned reflex activity; whereas before 
irradiation, similar or even larger doses of aminazine caused only some de- 
pression of the cerebral activity. This led Kupalov to the conclusion that 
the nervous system goes on acquiring other properties for a long time after 
irradiation and possibly even permanently. 

Electrophysiologic methods have yielded further evidence confirming 
what has been discovered about the radiosensitivity of the nervous system 
by the conditioned reflex method. 

In a series of papers published during the last few years, Livanov and 



MORPHOLOGIC AND PATHOPHYSIOLOGIC SIGNS 303 

co-workers (1959) and several other authors have described results obtained 
by using ordinary EEG and corticogram traces or from investigations per- 
formed with implanted electrodes. EEG's show that in rabbits and dogs 
expMDsed to whole body irradiation in large doses (about 1,000 r), the bio- 
electric activity and the irritability of the brain tissue are at first heightened 
and subsequently lowered. Hypnotic phases are also observed in various parts 
of the brain. 

Grigoryev (1958) found that variations in the EEG's of human subjects, 
taken during therapeutic exposure to x-rays, could be detected 18 sec after 
the beginning of the treatment. This effect occurred irrespective of whether 
the head, abdomen, or whole body was exposed to the x-rays. After the first 
exposures, however, the excitability of the nervous system was observed to 
increase: subliminal (3-irradiation) stimuli gave distinct physiologic effects, 
and the stimulation thresholds fell from between 6 and 12 sec to between 2 
and 4 sec. 

Further irradiation led to a drop in the responsiveness of the cortex. The 
number of paradoxical and ultraparadoxical responses to a strong light 
stimulus increased. Stimulation by caffeine, instead of ordinary activation 
of the electrical potentials, lowered the bioelectric activity; this indicates a 
lowering of the working capacity of the nerve cells. Toward the end of 
treatment, depression of the bioelectric activity usually set in, and the alpha 
rhythm became faster and more widely diffused throughout the cortex. The 
results of the first exposures were not stable and soon disappeared. Subse- 
quent exposures produced radiation effects which lasted much longer. Com- 
paring his findings with the dynamics of the excitation and inhibition 
processes discovered by other methods, Grigoryev (1958) says that after ir- 
radiation, the stimulatory process intensifies first, then diffuse inhibition 
occurs. 

On the theoretical level, Grigoryev's observation that vegetative rhythms 
appear in the cortex after irradiation, duplicating in frequency the cardiac 
and respiratory rhythms, is of distinct interest. In his opinion and in that of 
several other authors, the emergence of this rhythm indicates that the cerebral 
cortex is being more intensively influenced by excited subcortical formations. 

Geinisman and Zhirmunskaya (1953) and Grigoryev (1958) stress the fact 
that variations in the EEG are less pronounced after each consecutive x-ray 
sitting and regard this as a manifestation of the central nervous system's 
adaptation to repeated exposures by developing compensatory mechanisms. 
Nevertheless, along with the adaptation phenomena there cannot fail to be 
an accumulation of the variations occurring in the central nervous system 
under the effect of ionizing radiation. 

Grigoryev (1956) and Tsypin (1956) found that when rabbits were 
exfKDsed to whole body gamma radiation, even doses of 0.05-1.3 r caused 
distinct changes in the electric activity of the brain. In many animals the 



304 N. I. GRASHCHENKOV 

cortical responses were at first enhanced when tested by photic stimulation, 
but when the dose was increased to 7.8 r, cortical responsiveness and excita- 
bility diminished to the point at which there was no response at all to light 
stimulus. 

It follows from the research described above that even small doses of 
radiation energy (0.05 to 2, 5, or 10 r) are sufficient to cause detectable trace 
functional changes in the nervous system. The accumulation eflFect indicates 
that physiologic changes are at the root of this phenomenon. It can be as- 
sumed that even the natural level of radiation leads to similar changes, 
although this will overlap with repair processes in the nerve tissues. 

The changes in the nervous activity described, occur under the eflFect of 
whole body radiation and under local irradiation of the head (Meshchersky, 
1958; Fanardzhyan et al., 1960). 

The dynamics of the EEC's display certain characteristic features which 
make it possible to diflferentiate the reflex from the local responses of the 
brain to irradiation. 

Grigoryev (1958) notes that a lowering of the electrical potentials is ob- 
served following irradiation of the head. In whole body irradiation, the 
EEG response depends on the initial background, and the variations in the 
EEG are more generalized and as a rule localized in the opposite hemi- 
sphere to the one which is irradiated. 

With local irradiation of the temporal regions, variation in the electrical 
activity is observed within the irradiated zones of the brain or, at any rate, 
predominantly in these. We can therefore assume that the variations ob- 
served in the EEG's are both local and reflex in etiology. 

The considerable shifts observed in the interrelationship of the vegetative 
and endocrine systems as a result of radiation lesions are naturally leading 
investigators to pay attention to the influence of ionizing radiation on the 
functions of the hypothalamic and diencephalic parts of the brain and on the 
activities of the dififerent links in the vegetative nervous system. 

Work done in the laboratory of Livanov and Efremova (1957) has shown 
that 1 to 3 days after single whole body exposure (1,000 r), there is an 
abrupt change in the bioelectric activity of a rabbit hypothalamus. The 
variations in potential become more intensive, and there are recurrent spas- 
modic discharges in sharp waves. The response of the cerebral cortex to 
hypothalamic stimulation undergoes sharp variations and at times is even 
distorted. There is a lowering of the thresholds of the stimulations required 
to elicit the characteristic behavior responses (sniflfing, licking the lips). The 
rise in bioelectric activity is subsequently replaced by a lowering. 

Phase disturbance of the hypothalamic activity after mass irradiation of 
experimental animals has also been noted by Smirnova (1958) and Kon- 
drateva (1957). 

There is an increasing amount of evidence that other links in the vegeta- 



MORPHOLOGIC AND PATHOPHYSIOLOGIC SIGNS 305 

tue nervous system are also involved in radiation diseases. 

Klimovskaya (1958) noted increased lability of the preganglionic fiber of 
the cervical sympathetic nerve after irradiation. 

Nakhil'nitskaya (Lebedinsky's laboratory, 1959) demonstrated that the 
lability of the postganglionic sympathetic nerve is reduced after irradiation. 
This enabled Lebedinsky (1959) to suggest that "discoordination" of the 
different vegetative apparatus may play a considerable part in the genesis 
of responses to radiation. 

Disturbances or distortions of the vegetative responses from certain 
reflexogenic zones may constitute one of the major factors in the genesis of 
responses to radiation. 

Popova noted in 1954 that the reflex reaction of the blood process and 
respiration in response to stimulation of the gastric and rectal interoceptors 
of cats was first heightened and then lowered after the animals' heads had 
been exposed to 1,500 r. In 1954, Yaroshevsky noted a disturbance of the 
reflex leucocytosis under similar conditions. 

In 1957, Chernichenko, experimenting on rabbits, showed that exposure to 
600 r leads to a phase variation in the vegetative reflexes of the urinary 
bladder interoceptors. Komarov established in 1957 that the thresholds of the 
stimulation required for reflex heightening of the blood pressure were raised 
as a result of irradiation. 

A number of other authors have noted postirradiation variations in the 
pressor responses to adrenaline, a distortion of the response to Corazol 
(Metrazol) and lobeline, and a lowering of sensitivity in the chemoreceptors 
in response to a rise in the COo level in the blood. 

An observation of great interest was made by Kondrateva in 1957 in 
connection with the change in the character of the vasomotor and respiratory 
responses to direct stimulation of the hypothalamus after radiation, from 
which we may assume that the regulation of a number of physiologic proc- 
esses is distorted in the course of a radiation disease, precisely because of a 
disturbance of the reflex irritability of the hypothalamus. 

Another factor closely connected with functional disturbances of the cen- 
tral vegetative apparatus, and possibly even depending on its function, is the 
severe variations in the activity of the endocrine organs. In this connection, 
great interest attaches to the functional disturbances of the adrenal cortex 
noted by several authors, such as Tonkikh in 1958. 

Preliminary investigations performed in our laboratory, indicate that the 
cortical layer of the adrenal glands is involved in the response to radiation. 

As the scope of investigation widens in regard to both objects and 
methods, our knowledge of the influence of radiation on various parts of the 
nerv^ous system is steadily expanding. Today we have convincing evidence 
of the influence of ionizing radiation on the activity of the trunk apparatus 
of the nervous system and the cerebellum. 



306 N. I. GRASHCHENKOV 

Mushegyan and Abovyan showed in 1950 that radiation of the optic 
thalamus in frogs leads to characteristic inhibition of the spinal reflexes. 

In 1957 Yanson established that, after rabbits had been exposed to 1,000 r 
of x-radiation, a phase change began to occur in the condition of the labyrin- 
thine and cervical tonic reflexes. This change intensified during the first 2 
days but was unstable from the 3rd day on. During the first 3 days, a devel- 
opment of the plastic tonus in the extremities was observed and maintained 
for 7-10 days. 

Biryukov (1957) assumes that ionizing radiation acts on the reticular 
formations of the brain stem, basing this view on the similarity between 
certain physiologic effects produced by the action of aminazine and those 
produced by ionizing radiation in birds. 

Livanov and co-workers (1959) regarded the diffuse character of the 
EEG variations observed when the organism is exposed to ionizing radiation, 
as grounds for assuming that the reticular system of the brain stem must be 
included among the structures which condition the responses of the nervous 
system to radiation. The EEG was similar to that of a desynchronization 
response. Livshits, exposing the cerebellar region to directed x-rays, observed 
sympathetic phenomena similar to the responses elicited by direct stimulation 
of that organ. 

The influence of ionizing radiation on the function of spinal mechanisms 
is widely known from the literature, but new discoveries have been made in 
the last few years. Fedorova (1958) and Kudritsky (1957) have shown that 
even small doses of radiation (10 r) lead to a change in the time taken by 
the flexor reflexes of the posterior extremities of rabbits. When the rabbits 
were exposed to daily radiation doses approaching the permissible limits 
(0.1-0.5 r) for 14 days, the total activity (1.4-7.0 r) produced a lowering of 
the flexor excitability. After 37 exposures, the flexor response threshold 
ceased to alter, but its response was distorted when urethan was injected. 

Kudritsky (1957) showed that a change in the time taken by the flexor 
reflexes of the knee occurs mainly as a result of variations in the activity of 
the central apparatus and, to a lesser extent, of variations in the terminal 
apparatus of the reflex arc. 

Godin and Gorshkov (1957) note that in rats fed with small doses of 
radioactive sodium (0.5 [xc) the time of the defense reflex is slightly 
reduced, and the spread in the variations of this reflex is increased. When 
250 IXC were administered, the reflex time was first reduced and then began 
to lengthen. When the dose exceeded 300 fxc, the unconditioned reflex 
activity was immediately inhibited — an indication that the time taken to 
effect the reflex had increased. 

In peripheral nerve, the functional variations following irradiation are 
seen to follow a phaseal course. In 1934, Makarov noted parabiotic phe- 



MORPHOLOGIC AND PATHOPHYSIOLOGIC SIGNS 307 

nomena in frogs after prolonged exposure of an isolated nerve to beta rays. 
Vasilev (1957) observed the development of parabiosis in a nerve conductor 
after its exposure to alpha radiation. Bakin, Dolgachev, and Lomonos 
(1952) pointed out that not all the constituent parts of a peripheral nerve 
possess equal radiosensitivity. The excitability of the sensory parts is first 
observed to increase; later, sensitivity diminishes, and only after this has 
occurred do we find disturbance of the motor functions. 

Disturbances of the receptors and the consequent prolonged pathologic 
transmission of impulses from the periphery can play no small part in the 
nervous system's response to irradiation. 

Geinisman and Zhirmunskaya (1958) observed intensified transmission 
of impulses from the carotid sinus zone and the skin of a frog after these 
structures had been exposed to radiation. 

Tsypin (1956) noted a sharp intensification of the impulses reaching the 
nervous system from the eyes immediately after exposure to radiation. 

Zaretskaya (1956) noted phase variations in the impulses arriving from 
the chemoreceptors of the lymphatic ganglia of cats after exposure to 
radiation. 

Delitsina (1957) showed that after a rabbit's extremities had been exposed 
to 500 r there was at first a sharp intensification of impulses from the irradi- 
ated part, followed by subsequent weakening. 

Chernichenko (1957), by exposing the urinary bladder and one of the 
intestinal ansae to local irradiation, established that this caused a disturbance 
in the character of impulses arriving from the receptors of the aflfected 
organ. 

The fact that tolerance of radiation damage varies greatly from one sub- 
ject to another has led several research workers to pay attention to individual 
characteristics. It has been found that resistance to the effect of radiation 
depends largely on the type of higher nervous activity. Animals with well 
balanced higher nervous activity withstand ionizing radiation best; disturb- 
ances of the nervous activity due to the effect of radiation develop later in 
these animals, even after exposure to large doses. 

Lomonos (1959) notes that in dogs with a strong type of higher nervous 
activity, the conditioned reflex activity and the capacity to differentiate are 
maintained even on exp>osure to lethal doses of radiation. 

Khruleva (1958) showed that a temporary lowering, followed by a pro- 
longed heightening of the conditioned reflex activity, occurred in dogs with 
poorly balanced higher nei'vous activity when the animals were exposed to 
comparatively small doses of gamma radiation (50 r). In animals with a 
weak type of nervous system, such irradiation caused serious disturbances of 
the conditioned reflex activity, passing into prolonged neurosis. 

Similar results were obtained by Klimova (1957), Kurtsin (1957), 



308 N. I. GRASHCHENKOV 

Fadeeva et al. (1957), Kotlyarevsky et al. (1957), and other authors using 
animals exposed to various doses and types of radiation. 

The nature of the nervous system's response to the efifect of radiation 
depends also on its condition at the moment of irradiation. 

Grigorev (1956), by administering various drugs to a patient before x-ray 
therapy, showed that when the stimulatory process was intensified after 
caffeine, the EEG response became much more intensive. When Bromural 
was administered, variation in the electrical activity was barely noticeable, 
and the EEG response to radiation was even less pronounced after adminis- 
tration of quinine. 

Pomerantseva (1957) did not observe any radiation responses in animals 
exposed to radiation during sleep. Death caused by radiation after the 
animal had been awakened occurred a good deal later than in the control 
animals. 

Kazaryan and Saakyan (1960) describe how the severity of radiation 
damage is considerably reduced in animals in hypothermia. 

Soviet research workers have also studied the condition of the hematoen- 
cephalic barrier in animals exposed to radiation. 

Stern and co-workers have shown that as a result of exposure to ionizing 
radiation the resistance of the hematoencephalic barrier changes in two 
stages. After irradiation, the transition of various indicators from the blood 
to the brain, which can be noted only 45 minutes after irradiation, becomes 
more intensive (Stern, 1957, 1960; Gromakovskaya and Rappoport, 1957, 
1960; Goncharenko, 1960). Zaiko (1960) found that the peak increase in 
the permeability of the barrier occurred the 4th day after exposure with 
radioactive phosphorus (P^-). In the second phase the resistance of the 
barrier increases. 

Stern's co-workers noted that by administering neurotropic preparations 
(atropine, Novocaine, morphia), it was possible to alter at will the perme- 
ability of the barrier structures. This result at the same time confirms the 
fact that nerve factors play a part in the mechanism by which the barrier 
is disturbed. 

Arlashchenko (1955), using fluorescein as an indicator in investigating the 
condition of the hematoophthalmic barrier, showed that there is a sharp 
increase in permeability directly after exposure to radiation. 

It has been found that the degree of disturbance to the histohematic 
barriers is directly dependent on the dose of radiation (Arlashchenko, 1955; 
Goncharenko, 1960). 

There is every reason to assume that disturbance of the function of the 
histohematic barriers (in particular the hematoencephalic barrier) is one of 
the important basic factors in causing damage to nerve tissue exposed to 
ionizing radiation by making it easier for a number of alien and toxic sub- 



MORPHOLOGIC AND PATHOPHYSIOLOGIC SIGNS 309 

stances formed in the organism during irradiation to pass into the central 
nervous systein. 

Clinical Symptoms Appearing as a Result of Ionizing Radiation 

Much work has been done in recent years on the cHnical changes occur- 
ring in the nervous system in humans exposed to \arious types of ionizing 
radiation. 

Patients suffering from chronic radiation sickness complain first of tired- 
ness, torpidity, apathy, poor sleep, irritability, loss of memory, vertigo, 
nausea, and a tendency to weep; in other words, complaints of an asthenic 
nature are predominant in the first stage. 

The symptoms revealed by objective research arc predominantly those of 
vegetative dysfunction: lability of cardiac activity, increased activity of the 
vasomotor nerves, pronounced red dermographia, abundant sweating, in- 
tensification of the pilomotor refiex, acrocyanosis, and variation in the pulse 
rate and arterial pressure.^ Along with these symptoms of vegetative dys- 
function certain symptoms of damage to the somatic nervous system have 
also been identified : lowering of the refiex from the mucosa, Chvostek's sign, 
pseudobulbar signs, hypesthesia in the distal parts of the hands and feet, and 
lowering of the sensitivity to vibration. 

Trophic disturbances are also pronounced: brittleness and exfoliation of 
the nails, trichorrhea, hemophilia of the gums, and xeroderma. In more 
serious cases these symptoms are accompanied by pains in the extremities, 
distinct symptoms of sensory strain, disorder, nystagmus, and extrapyramidal 
disorders. 

Allowing for slight shades of difference, these symptoms indicate, in the 
main, the nervous system's response to all types of radiation (gamma rays, 
x-rays, radioisotopes, radioactive luminous compounds). The picture of 
damage to the nervous system is, accordingly, dependent on the close rather 
than on the properties of the type of radiation.- 

The basic factor determining the neurologic picture is vegetative disturb- 
ances. The forms of damage mentioned are regarded by some authors as 
caused basically by functional disorders of the higher vegetati\e centers 
situated in the hypothalamic region (Kurshakov, 1954: Kozlova ct al., 1957; 
Kozakevich, 1957; Shamova, 1958; Morozov ct al, 1957). This hypothesis 
is supported by the fact that pronounced vegetative trophic and endocrine 
phenomena are combined in the pathologic picture, as Kozakevich pointed 

' Disturbances of the oculocardiac reflex, attacks simulating Meniere's syndrome, 
and stenocardiac manifestations. Skvirskaya (1956) draws attention to distinct mani- 
festations of a regional spasm. 

■ Certain changes in the nervous system, however, can be detected even when the 
organism is exposed to small doses (Gusko\a. 1960). 



310 N. I. GRASHCHENKOV 

out. Moiseev (1957) detected serious morphologic changes in the hypophysis 
of guinea pigs after irradiation. 

Shamova (1958) demonstrated the distortion of several vegetative reflexes 
such as Scherbach's heat reflex, and variation in the sensitivity to ultraviolet 
radiation, which, with the clinical picture supports the hypothesis that the 
hypothalamus plays a leading part in onset of the disease. 

These findings are in full agreement with our own morphologic research, 
which also indicates that exposure to radiation causes pronounced damage 
to the hypothalamic region. Various authors suggest different systems of 
classification for the clinical symptoms that have been discovered. 

The early phase is described as moderately pronounced cerebral asthenia 
which manifests itself as an asthenovegetative syndrome. More pronounced 
changes are described as an astheno-organic syndrome (encephalopathy). 

There are also a good many dubious descriptions. Khazanov and Korenev- 
skaya (1958), for example, have described a case of neuritis of the femoral 
nerve as being due to the eflfect of radiation. 

The number of works devoted to acute radiation sickness is considerably 
smaller. Pigalev (1954) divides the disease into three phases: acute, sub- 
acute, and chronic. In cases of severe damage death occurs before the end 
of the acute phase. 

Kurshakov (1954) subdivides the acute form into four periods: first period, 
a few hours after exposure; second or latent period, lasting a few days to 
2 weeks; third period, grave symptoms; fourth period, recovery. 

It follows that ionizing radiation affects the activity of all links in the 
nervous system (the receptor apparatus, peripheral nerves, the peripheral 
parts of the vegetative nervous system, spinal cord, different apparatus of the 
brain stem and hypothalamic-diencephalic region), and it has a direct eff"ect 
on the cerebral cortex. 

It is difficult to determine which of these many interrelated parts is most 
affected, since the disturbance of any one leads to deviations from the 
normal condition in all parts of the nervous system. 

In my view, the most important damage done by ionizing radiation is to 
the receptor apparatus, the higher vegetative and endocrine centers of the 
superior parts of the brain stem and the cerebral cortex. 

One of the main effects of ionizing radiation on the receptor structures 
(resulting either from the direct action of radiation or from the action of the 
radicals formed in tissues during irradiation) is that the normal transmission 
of the nervous system's information is disturbed and several reflex responses 
are distorted. 

As the well known research by Vvedensky and Ukhtomsky has shown, the 
excessive inflow of impulses favors the development of parabiotic states and 
pathologic dominants at different levels of the nervous system. 

Functional and organic changes occurring in the hypothalamic and di- 



MORPHOLOGIC AND PATHOPHYSIOLOGIC SIGNS 311 

encephalic region and in the vegetative structures situated in this region 
also have great significance. 

In the conditions created as a result of exposure to radiation, the correc- 
tive activity of this part of the nervous system in relation to the various 
endocrine and vegetative structures becomes particularly important. Dis- 
turbance of this correlation or of the correct interrelationships between the 
diencephalic structures and the cerebral cortex will obviously bring about a 
whole series of vegetative and endocrine disturbances, such as are observed 
at all stages of radiation sickness. 

Functional disturbance of the higher parts of the brain depends on the 
direct influence of radiation on the nerve cells and on disturbance of the 
activity of the subcortex and receptor structures. 

Functional disturbance of the cerebral cortex has an effect on the charac- 
ter of the higher nervous activity and leads to the loss of one of the main 
functions of the cortex, namely, correct adaptation of the individual's reac- 
tions to environmental changes. 



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GENERAL DISCUSSION 

Percival Bailey (University of Illinois): I was happy to hear that the effect 
on the nerve cells can be primary and not secondary to the disturbance of the 
circulation. That I have always believed from long years of examining brains 
after irradiation for brain tumors and from results of work in my laboratory by 
Dr. Arnold and his associates with the effect of x-rays of high energy produced 
by the betatron. 

Webb Haymaker (Armed Forces Institute of Pathology): Dr. Bailey, I believe 
you mentioned that radiation "can" injure the nerve cell. I suppose you used 
"can" intentionally, rather than "may"? 

Percival B.mley: Naturally. Nerve cells can be injured also by disturbance of 
the circulation, and they are injured at times by disturbance of the circulation in 
brains that have been radiated. I believe there is also a primary effect on the 
ner\'e cells which is unrelated to the circulation. 

Carmine D. Clemente (University of California Medical Center): The problem 
of direct or indirect effects of radiation on the various elements in the nervous 
system is something that has plagued this field for a long time. I don't think we 
can quite let Dr. Bailey get away with what he has said without replying with 
some remarks. When we think about the radiosensitivity of the various elements 
in the brain we must talk about dosage. If one considers radiating an entire brain 
with high doses such as 5,000 r, 10,000 r, 50,000 r, or 100,000 r, that is one ex- 
periment, but if one considers the effects of brain radiation in the same tissue 
areas of maybe doses of 1,000 or 2,000 r, that is a completely different experiment. 
Furthermore, I believe that when a small part of the brain is radiated in com- 
parison to the entire brain it becomes a third experiment, and the dose-brain 
volume variable must be considered. When effects of high dosage radiation are 
described in the realms of 10,000 rad and 15,000 rad, it certainly is conceivable 
that neurons are directly affected. On the other hand, not only are the neurons 
affected, but the neuroglia and the blood vessels are equally affected. With these 
high doses, it seems to me that there is an analogy of taking a piece of brain and 
putting it in a frying pan, and thereby getting a lot of pathology in the tissue, and 
then saying "look at what's happened to the neuron." Let us think, however, of 
discussing the differential radiosensitivity of endothelial cells, glia and neurons, 
and discuss low dose effects. Under these circumstances an endothelial cell in the 
brain is a much more radiosensitive element than the neuron. 

Arthur Arnold (Chicago, Illinois): Dr. Clemente brought out the important 
point of total dose, namely dealing with 1,000 r or 10,000 r. .'\nother fact or 
equally important is dose rate. One can take a dose of 3,000 r and create an effect 
at 50 r per minute, and increase that rate to 200 r per minute and have an 
entirely different effect, not so much a qualitative effect, but a quantitative one. 
For example, I can take a monkey and deliver 10,000 r from a betatron through 
the frontal lobes in a single small beam, and it will lobotomize the animal, 

315 



316 GENERAL DISCUSSION 

providing I have given a dose rate of 200 r or more. If I reduce the dose rate to 
50 r then the effect is one-half or one-third as much. I think the factor of dose 
is important in responsiveness in the nervous system, and the total over-all effect 
depends on the dose as well as the dose rate. Dr. Innes brought out a feature that 
I thought was probably the highlight of the whole program, the problem of control. 
He showed in the rat that some of the animals, after a period of time, can develop 
neurolytic lesions in the field of demyelinization which probably are due to a virus. 
I have seen the same thing in rabbits and dogs, and would emphasize that, if you 
have to do a great deal of radiation studies, stay away from these animals. I have 
followed monkeys for as long as 8 or 9 years and have never seen such demyelinating 
disorders, which points to the monkey as the ideal animal for radiation studies 
on the nervous system. There seems to be a dilTerence in opinion as to whether 
we are dealing with direct or indirect effects and as to whether there is a total 
over-all effect from a massive dose; but I firmly believe there is a differential 
particularly when you are dealing with the glial cells and, through numerous 
studies on tumors, we know that there can be a differential response in a tumor 
cell. One would say that this is a different problem than the over-all nervous 
system; but there appears to be, in the adult nervous .system in the well developed 
animal, differences in responses between the types of glial cells as compared to, 
for example, the vessel structure. I have noticed that glial cells responded earlier 
than the vessels, and yet we have also noted that in time some of the late delayed 
necrotic effects arc obviously vascular, but the myelin changes may be related to 
all of those. 

Webb Haymaker: Professor Grashchenkov alluded to these delayed lesions, 
and he made a distinction between the vasomotor theory and another theory. 
Would you mind elaborating a little on that matter of the delayed radionecrosis? 
N. J. Grashchenkov (Moscow, U.S.S.R.) : It is not my personal investigation, 
but from my view, you have serious destruction of certain cells of the nervous 
system, like those of the subthalamus and hypothalamus. Some lesions were con- 
nected with other signs of destruction such as the spinal effects, caused by damage 
to cells of the spinal cord. Mainly, however, the damage is located in the thalamic 
tissue. 

Webb Haymaker: One point that Professor Schummelfeder brought out with 
respect to these late lesions was that in the literature it was suggested that they 
may represent a sensitive reaction in the vessels. I wonder if Dr. Leon Roizir 
would comment on, from what he has seen this morning of Professor Scholz's 
presentation, whether he sees anything in these vessels in relation to his studies 
on sensitivity reaction. 

Leon Roizin (New York State Psychiatric Institute): Orville Bailey has em- 
phasized the time factor in relation to delayed reactions, particularly as they were 
affecting blood vessels. He said the character of the lesions increased with time. 
Professor Scholz also emphasized the fact of certain delayed reactions. We have 
studied for several years in connection with experimental vascular encephalomyelitis 
by using as the main antigen total brain or brain fractions. The technique consists 
of brain or brain fraction suspensions emulsified in antigens and injected sub- 
cutancously or intramuscularly. After the injection there is a delay of from 3 weeks 



GENERAL DISCUSSION 317 

to several months. During this so-called latent period of sensitization, the animals 
show a mosaic of neurologic symptoms. Some who are sacrificed or die during 
the acute phases showed edema and vascular changes of the necrobiotic or in- 
flammatory coverings around the veins. Of particular interest to me was the reac- 
tion which Professor Scholz showed in the H and E preparations and with the 
elastic stain. He emphasized the fact of so-called plasmodic or fibroid material 
which has involved the vascular material as well as necrosis. We have observed 
similar changes during the early phases of the allergic reaction in experimental 
encephalomyelitis. It occurs to me that possibly the x-ray effects on the tissue 
produce a necrobiotic phenomena; and this phenomena, which requires a certain 
period of sensitization, initiates a chain of reactions with particular involvement 
of the blood vessels. I would like to suggest the possibility that delayed reactions 
could be produced by a necrobiotic process, induced by irradiation. 

J. R. Innes (Brookhaven National Laboratory): I am pleased to hear Dr. 
Arnold mention that he thought the monkey was the ideal animal for radiation; 
however, although they don't suffer the legion of disorders which affect the ner\'ous 
system of man, Van Bogaert, Schair, and others, show that they do suffer a 
remarkable variety of neurologic diseases, some of them unique. Curiously, some 
show lesions like the ones I showed and Dr. Scholz showed in his rabbits, which 
are specific in monkeys, except the gorilla and chimpanzee. In any work involved 
with monkeys where they are kept a long time, you must be alert to neuroparalysis, 
palsy, blindness, and leucoencephalomyelosis, which although uncommon is a well 
recognized condition. In this disease, brain as well as spinal lesions appear scat- 
tered, spotty, and more denominational. Concerning the specific effect of irradia- 
tion on the myelin system, I think this effect on the neuron is specifically on white 
matter and far more than on the axis cylinder. In the cord it seems to affect the 
ventrolateral more than the dorsal. Let us remember that there is such a thing as 
a conducting cylinder, the myelin system. 

Percival Bailey: It is true that monkeys have diseases of the brain, like other 
animals. But on our animals we never saw anything of the kind. Furthermore, it is 
not possible that any such disturbance could have occurred in them, because we 
used a beam produced by the betatron which makes a constant lesion without any 
scatter, and in one hemisphere using the opposite hemisphere for the same animal 
as a control. 

Webb Haymaker: We would like to hear from Dr. Alvord because of his 
sensitivity studies with the granular layer of the cerebellum under different 
physiologic conditions. 

E. C. Alvord (Seattle, Washington): The answers we get usually depend on 
the methods we use. I would like to develop the idea that there is specificity within 
the nervous system in its susceptibility to the effects of radiation and to bring up 
for comment the irreversibility or reversibility of some of these changes and the 
site of swelling or edema. When a large part of one cerebral hemisphere has been 
removed and a guinea pig is given 7,500 r in the head, the normal guinea pig 
will die in 24 hours. If you provide internal decompression by surgery, it can 
live 4 to 6 days. Those sacrificed 4 hours after irradiation look reasonably normal. 
The others show swelling of the entire brain, with cerebellar consular herniation. 



318 GENERAL DISCUSSION 

By comparing the wet and dry weights of the cerebral hemispheres, brain stem, 
and cerebeUum, we see that the accumulation of water is localized to the cere- 
bellum and that the cerebral hemispheres, operative or inoperative side, are such 
that the brain cell shows practically no swelling. With this prolonged time it 
becomes obvious that in the cerebellum the granular cells go much further than 
we had thought in the normal guinea pig. After 5 days, there is karyorrhexis, as 
well as pyknosis, but there is no evidence of reversibility. I would ask Dr. Vogel 
if his remarkable, almost inconceivable, recovery of the monkey at 72 hours might 
not be a reflection of the individual variation in sensitivity of monkeys to irradia- 
tion near the threshold level, probably around 10,000 r and that the monkey that 
showed no change at 72 hours really didn't show any 48 hours earlier. 

F. Stephen Vogel (Cornell Medical Center): The graph that was shown with 
the spectrum of changes was from a group that were all exposed to 10,000 r. It 
was felt that this dose was capable of regularly producing pyknotic changes in the 
granular cells. Someone could postulate that any animal sacrificed at one time or 
another had either shown this change or would show it at the time of sacrifice. 
The pairs of animals were killed so that we had not one animal at 72 hours, but 
four animals. I think similar findings have been seen in the rabbit in our labora- 
tory and elsewhere, coupled with the fact that there was no evidence of inflamma- 
tion or karyorrhexis later nor evidence of pyknosis or decrease in number of cells. 
I think one is justified in suspecting that these four animals showed the same type 
of change as the much larger group of about 20 animals that were killed earlier. 
With the observation that in tissue culture this change appears to be transitory, 
we feel that this is a difference in sensitivity. I think one would have to increase 
the dose above 30,000 r, perhaps, before one reached the stage where you could 
destroy the granular cells of the monkey. 

L. M. H. Larramendi (University of Illinois): In using the sciatic nerve of the 
bullfrog, I assume the action potentials that Dr. Gaffey showed were of the A 
fibers. I would like to know if he has tried the same experiment to the B and C 
fibers. Anatomically they are different. And I think Dr. Grashchenkov mentioned 
that the autonomic nervous system is more sensitive, and the B and C fibers are 
related to the autonomic system. Concerning previous comments by several persons 
about the granular layer of the cerebellum, they were referring continuously to 
the granules of the granular layer of the cerebellum. The implication I think was 
that all the granular cells were within the granular layer of the cerebellum and 
that they are neurons. This is not the case. 

C. T. Gaffey (Donner Laboratory, University of California, Berkeley, Cal- 
ifornia): Concerning the species of frog, the Rana catesbeiana is the bullfrog, and 
the Rana pipiens is the grasshopper frog. A question was raised concerning the 
sensitivity of A, B, and C fibers. It is my understanding that the Rana catesbeiana 
does not have B fibers from a histologic point of view. Gerstner has studied 
sensitivity to x-irradiation within the A fibers (American Journal of Physiology, 
1956). I believe the A-gamma fibers have essentially the same sensitivity, but 
the B fibers are more resistant. Most of the changes are rather minor between 
these fiber types in the A group. He also thought the C fibers were too thin and 
delicate. 



GENERAL DISCUSSION 319 

James Lott (North Texas State College): Since Dr. Gaffey's results seem to 
not coincide with Dr. Bachofer's and mine, working with peripheral nerves in the 
rat, I would like to ask him about some of his techniques. I would like to know 
how often he stimulated the nerve during the control period, how long after he 
removed the nerve did he begin his recordings, how often he stimulated the nerve 
during irradiation, when he was taking his recordings, and whether he has ever 
seen an increase in the action potential due to alpha or deuteron irradiation. 

C. T. Gaffey: The stimulation period of the frog was short. About half a 
minute to a minute. Once every 2 hours the nerve was monitored. So, essentially 
there would be no change in the nerve due to the stimulation effect. It is known 
that stimulations at low frequency, compared to high frequency, will change the 
magnitude of the potential of the compound A fibers. These stimuli that were 
given were maximal for a response. Enhancement of neural output is demonstrated, 
but there is some confusion in the season of the year in which the enhancement 
is shown. In winter frogs, enhancement is strongest. In summer and fall frogs, 
enhancement is minor. It would appear that the potassium gradients across the 
fibers are seasonal and reflect the degree of enhancement shown in any par- 
ticular experiment. 

Orville T. B.mley (University of Illinois): The pathology of radiation in the 
central nervous system leads to a series of questions which are very difficult to 
separate and on which we have conflicting opinions which may be related to 
extraneous factors. In regard to the long latent period, I am perhaps somewhat 
influenced by experiences with radiation changes in skin. Experience with these 
makes this long latent period more understandable. One patient seen over many 
years with repeated biopsies was an eminent scientist who, as late as 35 years 
after he stopped using x-ray, was not only developing new carcinomas but was 
developing patches of typical x-ray dermatitis in previously normal skin. The 
participation of allergic responses in the pathology of radiation is an open ques- 
tion. Similar end results do not necessarily mean similar pathogenesis. Because the 
reconstituted vessels in repair have the same appearance when these processes 
are finished that they have an allergic arteritis does not necessarily mean that all 
the stages or that the pathogenesis is the same. Finally, one of the strongest argu- 
ments for differential effects of radiation on the various types of cells in the central 
nervous system is the alteration in the sequences of histologic repair. 



CHAIRMAN'S SUMMATION 

Webb Haymaker: We have heard a good deal about the cerebellum. One of 
the interesting observations pointed out by Dr. Vogel was with reference to the 
cerebellar cells in his tissue culture. Granular cells underwent significant changes 
in response to radiation, but the inner cells of the cultures apparently were un- 
affected. This must mean that granule cells are more radiovulnerable than other 
cerebellar cells, as we already know. The point of Dr. Vogel's observations is that 
nuclear pyknosis and early enlargement of the cytoplasm, pointed out also by 
Dr. Schummelfeder, took place without the benefit of circulatory disturbance. 
In his presentation on the cerebellum of the mouse. Dr. Schummelfeder considered 
the effect of circulatory disturbances on granule cell changes, and at all doses 
between 60,000 r to 2,000 r granule cells were always altered first. Then after a 
latent period, the vessels underwent morphologic changes. To him, and apparently 
to the two Drs. Bailey, quite definitely the ultimate pathologic picture when the 
latent period has passed is apparently the outcome of the combination of direct 
effects and the effects of vascular origin. The paper by Drs. Hager, Hirschberger, 
and Breit aroused particular interest because it dealt with observations under 
electron microscopy. They too, found that the granule cells of the cerebellum were 
particularly radiovulnerable, but they dealt also with the broader aspects of the 
problem of pathogenesis. This area of pathogenesis is a kind of twilight zone 
without a horizon, where observation still blends too much with opinion. Dr. 
Hager and associates, using high doses of 40 kv x-rays, provided the following 
timetable according to electron microscopy as seen in the brain of the Syrian 
hamster: First, there was increased vascular permeability as manifested by swelling 
of cells which they called astrocytes, by plasma exudation, and by erythrodiaped- 
esis. Their observations showed that at these dose levels the first stage seen in the 
electron microscope was vascular disturbance. Dr. Hager and his associates feel 
that the loosening of tissue and the fine sponginess seen after radiation is merely 
a reflection of the accumulation of fluid in the astrocyte, or what has been called 
the astrocyte, and that gross sponginess is attributed to astrocytic and other mem- 
branes either in vivo or during tissue processing or both. These cells called 
astrocytes become watery. At the same time that the astrocytic change occurs, they 
said, the capillary endothelium shows suggestive swelling, and about this time a 
structureless material considered to be plasma exudate is seen in the perivascular 
space. Only later are nerve cell changes seen, and still later changes are seen in 
the large blood vessels. Dr. O. T. Bailey emphasized the relative radiovulner- 
ability of the astrocyte. Professor Scholz and his associates demonstrated with 
high dose x-rays that astrocytic processes break down at an early stage, at a time 
when only minor changes are visible in nerve cells. I think we are now at the stage 
of knowledge at which we can generalize and say that when radiation upsets the 
transport in the metabolic mechanism of the astrocyte, nerve cells are bound to 
suffer as a consequence. Dr. Innes and Dr. Carsten showed us the later stages of 

321 



322 CHAIRMAN'S SUMMATION 

what happens after radiation. One of the interesting points was the variable latent 
period, namely, anywhere from 3 to 9 months after the animals were exposed 
to 3,500 r, before lesions became apparent. This matter of the latent period, as 
it concerns individual variation, is a common problem, and it always requires a 
large series of animals before one can reach dependable conclusions. This brings 
us to the concept of the delayed radionecrosis described by Professor Scholz. He 
demonstrated swelling and disintegration of vessel walls before there was any 
visible change in the surrounding spinal cord substance. The question arises as 
to whether these vascular changes and exudative phenomena, the existence of 
which there can be no question as seen in Professor Scholz' lantern slides, may 
be the outcome of some peculiar sensitivity reaction. It is interesting that Dr. 
Roizin is of the opinion that what he saw today might possibly be construed as 
a sensitivity reaction and that the change was primarily in the vessels rather than 
in the nerve substance around the vessels. Dr. Vogel pointed out, as did Dr. 
Schummelfeder and others, that radiovulnerability of the cells in the central 
nervous system varies with the species. This is a general principle of which we 
must all be aware, but which some of us now and then tend to overlook. 



PART III 

Particle Irradiation of the 
Central Nervous System 



The Use of Accelerated Heavy Particles for 

Production of Radiolesions and Stimulation 

in the Central Nervous System 

Cornelius A. Tobias* 

Medical Physics Division, Donner Laboratory, 
University of California, Berkeley 



The central nervous system has a more intricate ors;anization than any of 
the other organ systems of the human body: its functions seem to depend on 
the spatial configuration of its delicate structural elements, and on spatial 
and temporal interrelationships of its neuronal discharges. 

Through past centuries up to the present, a tremendous amount of knowl- 
edge has accumulated with respect to gross, microscopic, and submicroscopic 
anatomical structures of the central nervous system ; and relatively recently 
it has become {X)ssible by the use of surgical instrmnents to make lesions in 
various parts of the brain, then follow^ the abnormal pattern of physiological 
and electrical activity thus created by a variety of techniques. In surgical 
interference with brain structure, whether this be done for the purpose of 
physiological study or with therapeutic aim. serious limitations are encoun- 
tered: the entire path of the surgeons needle or knife produces injury from 
surface to depth with interruption of neuronal pathways and blood vessels. 
The result can be hazard for hemorrhage, subsequent necrosis, and later 
formation of scar tissue along the pathway of surgical injury. The presence 
of scarring by itself can lead to disturbances of electrical fimction and to new 
injury with the production of more extensive lesions. While there are con- 
stantly new improvements in operative technicjue and while much new 
knowledge is gained by the current methods, one should maintain an interest 
in novel approaches to the problems of neurophysiology. The role of ac- 
celerated heavy particles will be outlined here in historical background. This 
is done to illustrate that man's curiosity in relatively remote and discon- 
nected, "useless" aspects of nature can sometimes lead to new tools, new 
methods, and new knowledge in very practical and useful realms. 

* The author's research is supported by the .\EC. 

323 



326 CORNELIUS A. TOBIAS 

Acceleration of Atomic Nuclei 

The original reason for artificially accelerating nuclei of light atoms was 
the realization that such particles might penetrate the atomic nucleus. About 
the same time that Ernest Lawrence built the first cyclotron (see Lawrence 
and Livingston, 1932) for acceleration of deuterons, protons, and alpha 
particles, it was realized by Zirkle (1932) that atomic nuclei, particularly 
alpha particles, were more efHcient in killing cells than x- or gamma rays, 
and that the major effect of alpha particles was on cell nuclei. Acceleration 
of heavier nuclei (e.g., lithium, mercury) was attempted in the early 1930's 
(Sloan, 1935), but did not become practical until about 1940 (Alvarez, 1940; 
Tobias and Segre, 1946), when the nuclei of carbon were accelerated in the 
Berkeley 60 in. cyclotron to about 120 Mev kinetic energy. In about the 
same year, the Swedish scientist Edlen (1941, 1942) discovered that multiply 
charged atoms of various elements, including carbon, calcium, and iron, 
were constantly present in the solar corona. These elements are either a result 
of emissions of solar plasma into surrounding space, or come about by inter- 
action of the interplanetary dust with solar radiations. In 1948, Freier et al. 
discovered in observations taken on photographic emulsions in high altitude 
balloons, that primary cosmic rays reaching the earth at the top of the 
atmosphere were protons, alphas, and heavier nuclei, many of them with 
billions of electron-volts kinetic energy. It was demonstrated that at least 
part of the heavy nucleons arriving at the earth originate in the sun or solar 
corona. 

For 30 or 40 years physicians concerned with tumor therapy realized the 
desirability of concentrating radiations in limited regions in the body. High 
energy x-rays, gamma rays, and neutrons were tried in succession. In 1946, 
Robert Wilson, then at the University of California, realized that high 
energy protons, due to their particular ionizing properties, might be useful 
for irradiation of deep seated tumors in the body. In 1948, with completion 
of the first large cyclotron, the Berkeley group undertook systematic investi- 
gation of the usefulness of protons and deutrons in biological research. 
Tobias et al. (1952) demonstrated that high energy particles (190 Mev 
deuterons) are useful for production of localized radiolesions in the body, 
and that the Bragg ionization peak for tumors in mice does indeed possess 
some advantages. A technique was developed for hypophyseal radiation of 
the rat (Tobias et al., 1954; Van Dyke et al., 1959) and monkey (Simpson 
et al., 1959), which led to a well-defined, chronic hypophysectomized state 
in the animals. In 1955, encouraged by the mounting evidence for hormone 
dependence of many human cancers and by the initial successes of surgical 
hypophysectomy (Luft and Olivecrona, 1953), and following initial studies 
of hypophyseal radiation of dogs with advanced mammary carcinoma (un- 



LESIONS WITH ACCELERATED PARTICLES 827 

published), investigation of human hypophyseal proton radiation was begun. 
Today more than 100 patients have had pituitary radiation (Tobias et al., 
1958; Born et al., 1960). Many cases of advanced metastatic mammaiy car- 
cinoma and a few of prostate carcinoma, advanced diabetes mellitus, acro- 
megaly, malignant exophthalmos, leukemia, and Cushing's disease were 
treated, and it appears that the hypophyseal radiation is useful in producing 
some regressions and in allowing studies of hormonal aspects of the abov^e 
mentioned diseases. The radiation is so well concentrated in the pituitary 
that only occasional mild secondary neurological effects are encountered. 

Several years ago it was also realized that accelerated particles made the 
production of lesions in the brain possible, so that the Berkeley group has 
initiated some studies on the hypothalamic radiation syndrome (Anderson 
et al., 1957). It was found that for production of radiation lethality the 
hypothalamic region is more sensitive than either the cerebral cortex or the 
pituitaiy. The hypothalamic lethal syndrome is a symptom complex inte- 
grated from failures of a number of homeostatic regulatory functions and 
often results in abdominal or intestinal bleeding as a cause of death. It was 
also shown that lesions in the region of the median eminence lead to diabetes 
insipidus, glycosuria, thyroid abnormality, and delayed sexual development. 
Roberts, Thorell, and associates (1957) demonstrated that a lesion in the 
posterior hypothalamus, not identical with the "appetite center," leads to 
decreased rate of growth and degeneration of eosinophilic cells in the 
pituitary. An interesting use of the Bragg ionization peak of alpha particles 
was made by von Sallmann et al. (1955) who selecti\ely irradiated the lens 
of the rabbit eye and determined the relative effectiveness of alpha particles 
and deuterons on lens epithelium for producing cataracts. 

In 1955 and 1956, the author and his colleague, Victor Burns, ^ had the 
opportunity to collaborate with a team at the University of Uppsala, at a 
time when their proton synchrocyclotron was adapted to tumor studies and 
neurologic irradiations (Larsson et al., 1956). Larsson and his associates 
developed a method for production of cutting "knife edge" lesions with a 
narrow beam of 185 Me\' protons which were passed through a narrow slit 
made of absorbing material. Knife edge lesions of the spinal cord were studied. 
By the use of a 1.5 mm wide beam on a rabbit spinal cord, it w'as possible 
to produce a sharply limited lesion with only minimal hemorrhages (Larsson 
et al., 1958; 1959). Rexed et al. (1959) produced cutting lesions in the 
upper anterior part of the rabbit brain with doses of 20,000 rad and found 
destruction of myelin sheaths, axons, and nerve cells. The lesions in the first 
3 months were confined to the region irradiated. Working with pigeons, 
Fabricius and Larsson (1959) are in the process of studying the localization 

' Present address: Biophysics Department, Stanford University. 



328 CORNELIUS A. TOBIAS 

of control of instinctive behavior by production of lesions that decerebrate 
the animals. In 1959, Leksell, who has pioneered in the applications of radia- 
tions to brain surgery, and the Uppsala team initiated proton neurosurgical 
applications by producing bilateral cutting lesions in the frontothalamic 
bundles in a patient with schizophrenia (Leksell, 1961). Sourander and 
associates (Andersson ct al., 1961) have supplied very interesting neuro- 
histopathological studies. 

While studies with deep lesions were in progress with high energy nucleons, 
another group of approaches took place with particles of a few million volts 
per nucleon. Pollard (1953) and associates have used protons for basic 
studies of enzyme molecules and phage particles. Zirkle and Bloom (1953) 
applied a microbeam of protons to partial cell irradiation. Soon after the 
nature of cosmic ray primaries were known (Freier et al., 1948) speculations 
arose that heavy primary cosmic rays could exert great radiobiological effects, 
particularly in the brain, where it was believed no recovery from radiation 
effects would take place (Schafer, 1950; Tobias, 1952). The first exposure 
of living cells to carbon particles was done in 1952 at the cyclotron (Sayeg 
et al., 1959), and an increased biological effectiveness of heavy ions on yeast 
cells was demonstrated. In 1957, both at Berkeley and at Yale University new 
accelerators became available which can deliver particles up to argon nuclei 
with about 10 Mev energy per nucleon, and detailed information is becom- 
ing available on the manner of action of these radiations (Brustad, 1960; 
Hutchinson, 1960). The chairman of this session and his associates (Malis 
et al., 1957) showed that a monoenergetic beam of protons can produce 
in animals "laminar" lesions in the brain only about 80 microns thick, run- 
ning quite closely parallel to the exposed brain surface. Malis et al. (1961) 
are presenting new data here. Haymaker and associates have traced the 
nature of pathological development of such radiolesions with alpha particles 
(Janssen et al., 1961). Van Dyke et al. (1961) have pointed to the great 
variation of radiosensitivity of the brain among different species and demon- 
strated the vulnerability of the blood-brain barrier by showing that fluores- 
cent dyes can penetrate the irradiated region early in the course of lesion 
development. It was also shown that very high doses in rat cerebellum with 
low energy beams can lead to sharp surface lesions with scarring virtually 
absent. 

Today, accelerated particles can be used for the production of a variety 
of well-defined radiation lesions. Work is in progress not only on the involved 
steps that lead to the development of radiation-induced necrosis, but also the 
accurately placed lesions are being utilized to obtain answers for many 
pressing problems. The role of histologically defined neuronal zones in the 
gray matter, anatomical studies of retrograde degeneration, the problem of 
regeneration, and that of the origin and nature of the electrical function of 



LESIONS WITH ACCELERATED PARTICLES 329 

the brain (Tilsjar-Lentulis and Tobias, 1959) are only examples of studies 
currently active. 

One of the major tools of the neurological research worker is electrical 
stimulation. We have shown during the last few months that radiation, too, 
can be used for stimulation of nerve activity. For some years it has been 
clear that specialized structures, such as the retina, can be stimulated with 
penetrating radiations. Hug (1960) only a year ago demonstrated that ten- 
tacles of the snail can "feel" the presence of x-rays. Conard (1956) has 
studied the effect of radiations on contractions of parts of intestine in vitro. 
In our laboratoiy (Tobias et al., 1961) pulsed beams of alpha particles were 
used on the cornea to elicit the so-called "corneal blinking reflex." It is clear 
that deep stimulation of structures of the central nervous system by heavy 
ion beams is a distinct possibility. Since the diameter of such beams can be 
made very small, and pulsing of the beams can be accomplished in various 
ways, a distinct possibility exists that in the future one might be able to 
scan limited regions of the brain surface with a deflected beam of penetrat- 
ing particles, delivering a "message" to certain cells in the brain by a pre- 
arranged spatial and temporal code, without the trauma of surgical 
intervention. 

Physical Techniques of Producing Lesions 

The physical characteristics of accelerated nucleons have been described 
in detail (Tobias ct al., 1952; Larsson et al., 1956; Birge et al., 1956; 
Brustad, 1961). Here we wish to give only an outline of those properties of 
radiobiologic importance. Usually an attempt is made to produce an almost 
parallel beam of particles in an accelerator. This task cannot be accomplished 
perfectly because in the course of acceleration in a cyclotron, radial and axial 
oscillations occur; in a linear accelerator there are also "defocusing" effects. 
In a linear accelerator it is possible, with the aid of electromagnetic focusing, 
to have almost all particles emerge from the machine in a single spot of 
perhaps 1 mm in diameter and within 0.1% monoenergetic. In the cyclotron, 
the particles emerge from a nonhomogenous magnetic field, and they are 
not parallel. In this instance one may focus them to obtain a slightly con- 
vergent or divergent beam, and one usually applies a slit system to select 
particles traveling nearly parallel. This way beams of about 0.1 degree 
angular divergence can be obtained, but a great part of the beam which does 
not satisfy the criteria of parallelity is not actually used. 

The monoenergetic particles penetrate nearly the same distance in tissue. 
Figure 1 gives the range energy relationship in water for protons, alpha 
particles, and for heavier ions as a function of the kinetic energy per 
nucleon. Also, on this figure are indicated the actual energy and range values 



330 



CORNELIUS A. TOBIAS 



0.1 



Al, g/cm^ 
1 10 



100 



1000 



10' 



10- 



c 
o 
_2 

w 

3 
C 

> 
0) 

o> 
a> 

® 10 

0) 



o 



ARGON 

NEON 

CARBON 

PROTON & ALPHA 

DEUTERON 



.^^J 




PROTONS, BERKELEY 



900mev ALPHA 
•^ -''^450mev 



DEUTERONS 

BERKELEY 



HILAC 
BERKELEY 




0.01 



100 



1000 



Grams/cm' 



Range in water or soft tissue 

Fig. 1. Range energy relationship for protons, alpha particles, carbon, neon, and 
argon nuclei. Black dots indicate ranges of the cyclotron beams available in Berkeley. 
Black triangle signifies the protons of the Uppsala cyclotron. Black square is the 
proton beam of the Harvard and French Atomic Energy Commission cyclotrons. Open 
circles indicate beams of the Yale and Berkeley heavy ion accelerators. 

which have been utilized at various laboratories in biological and medical 
studies. Since biological experimentation has usually been secondary to the 
use of machines in physics research, we may note that certain interesting and 
desirable energy ranges have not as yet been explored with biological speci- 
mens. For example, one does not have ions heavier than alpha particles with 
kinetic energy greater than 10 Mev per nucleon. Unlike x-rays or electrons, 
nuclear particles of equal energy travel almost to the same depth in an ab- 
sorber. Some particles are, however, lost because of inelastic collisions. Others 
show slight differences in their respective ranges owing to straggling and 
multiple elastic scattering. The root mean square fluctuation ^/AR' of the 
range R increases as the particle energy and penetration increases. 

V ^ (observed) — - ^R (initial energy spread) r Ar~ (straggling) r ^B (scattering) 

The exact relationships are complicated. However, the fractional straggling 

>/Aij^ for particles of the same velocity is independent of charge and varies 
with the square root of the mass of the particle. 



VAi 



R. 



Ri 



M, 



Va/ V M, 

2 

Thus, a proton has more than 40 times less straggling than an electron; 



LESIONS WITH ACCELERATED PARTICLES 331 

a heavy particle, e.g., oxygen has 4 times less straggling than a proton. For 
a given range, experimentally minimum range variation is achieved by 
making A '•* (initial energy spread) as Small as possiblc. Thus ideally, one would 
wish to accelerate particles with the proper energy for each range used in 
order to have optimum ionization properties. 

As a parallel beam of particles enters an absorber, due to multiple elastic 
scattering there will be a radial spread in the beam. Having crossed a dis- 
tance X in an absorber, the mean square radial distance r~ from theoretical 
straight line trajectory is 

7 rr 1/3 ^ Z^ 
where 6" is the mean square of the angular spread (in radians) 

where T is the kinetic energy, Z the atomic number of the absorber, p is its 
density, z is the atomic number of the fast charged particle, and a c a con- 
stant that depends on the allowable elastic scattering angles 6. For the same 
velocity a lighter particle has a longer range and vmdergoes more scattering 
than a heavier one. Also, the root mean square angle of scattering is propor- 
tional to the rate of energy loss; thus at high energy, where the rate of energy 
loss is low, there is less scattering than at low energy; the radial spread of the 
beam will be less if an absorber of high atomic number is vised. After a beam 
has emerged from an absorber, to the air, the spread of the scattered beam is 
much more noticeable than in a solid absorber, since in air there are greatly 
reduced numbers of collisions. The energy transfer to the absorbing medium 
along the path of an ionizing particle has been calculated by Bohr (see Evans, 
1955). Over a wide range of energies the rate of energy loss is almost in- 
versely proportional to the kinetic energy T of the particles {T'^'') ; thus 
low energy particles ionize much more heavily than high energy ones. The 
relationship breaks down near the end of the range where the particles pick 
up electrons as they stop, and also at very high energies, near 1 Bev per nu- 
cleon, where relativistic effects appear. Comparing protons to heavier acceler- 
ated particles of charge z, we find that at the same velocity the heavier particles 
transfer more energy to the medium, in proportion to z^. The rate of energy 
loss of various individual particles is plotted in Fig. 2 as a function of their 
kinetic energy per nucleon. Unlike x-rays, which show great dependence of 
energy transfer on the atomic number of the absorber, heavy accelerated parti- 
cles interact mostly with atomic electrons; thus the stopping per electron is 
nearly the same for all elements. For accurate dose calculations it is necessary 
to know the stopping power of the tissue, relative to nitrogen or other gas, 
where the ionization is measured. While more experimental work needs to 
be done in this field, some stopping powers are accurately known (Tobias 
et al, 1952). 



332 



CORNELIUS A. TOBIAS 



5x10" 



• 10^ 





^\ 


1 1 
\yArgon 

Carboivv \Neon 




^ 


\^ ^\Alpha 


- 


Proton, 


Deuteroivv ^^■>-.,.^ 

1 1 



10 100 1000 

Energy, Mev / nucleon 



Fig. 2. Rate of energy transfer from various particles to water as function of the 
energy per nucleon. Rate of energy loss in soft tissue is within 2% of this value. 



An actual beam of nearly monoenergetic particles ionizes along the so- 
called Bragg ionization curve, usually arrived at experimentally. This rela- 
tionship takes into account all phenomena of scattering and straggling. 
Figure 3 shows the Bragg ionization curve as measured for 40 Mev alpha 
particles for the purpose of the work reported at this conference with 
Haymaker ct al. Such a beam can cause laminar neurological lesions. In 
order to understand the way such lesions originate, Fig. 3 also contains a 
hypothetical dose-efTect relationship of the multiple hit type. It is easy to 
understand that pathologically visible lesions occur only in regions where 
the dose, as represented by the Bragg ionization curve, exceeds a certain 
threshold value. 

Experimentally the ratio {\/^:^R)/R is between 1/40 to 1/60 in the 
energy range of 10 Mev/nucleon to 200 Mev/nucleon. The lateral spread of 
the beam is such that it is possible to maintain a lateral variation of {\/r-)/R 
somewhat less than the magnitude of the range \ariation (\/A^/?)/i?. For 
an alpha particle beam of 10 Mev/nucleon the range is about 1300 fx, 
in tissue; the width of the Bragg peak is about 100 /x and the lateral spread 
of the beam in 1300 /x distance is about 10/x; for 190 Mev deuterons the 



LESIONS WITH ACCELERATED PARTICLES 



333 



20000 



M 

O 

^^ 10000 



o 

o 



ALPHA PARTICLES 
10.4 ± 0.2 Mev / amu 



0.5 1.0 

Depth, Millimeters 










^■- 



r 



Effect 



1007. 



^Tissue surface A Laminar lesion 

Fig. 3. Depth dose distribution for 40 Mev alpha particles. On upper right is a 
hypothetical dose-effect relationship. .'Xssumino that effects grt'ater than 50''r are 
visible under the microscope, the lower cur\e gives profile of the "laminar lesion." 



range in tissue is about 14.5 cm. the width of the Bragg peak is about 4 mm. 
and the mean lateral spread in the entire range about 3 mm. Figures such as 
these gi\e an indication of depth and minimal practical lesion size of the 
particles in Cjuestion. A great deal of experimental work remains as yet to 
be done to explore the limitations of the technique. It is practical to use 
knife edge lesions of 1 mm in diameter to a depth of about 4 cm in the 
brain: as the beam penetrates deeper, however, the lesion becomes less and 
less definite. 

The cross-sectional area of the beam may be shaped by metal apertures of 
thickness greater than the range of the beam. Usually, one monitors the 
beam with ion chambers as it emerges from the accelerator and calculates 
the ionization as function of depth in tissue from the known stopping power 
and composition. Experiments with phantoms are possible. 

An illustration of the relati\ e lack of scattering is shown in Fig. 4. Here a 
beam of 190 Mev deuterons, 1 X 2 mm in cross section, was passed through 



334 CORNELIUS A. TOBIAS 



i 



V 




Fig. 4. Photograph of head of rat which received 20,000 rad of deuterons in a 
small beam 1 X 2 mm in diameter passing laterally through the head. White spot in 
hair indicates passage of beam. Entry and exit spots are indistinguishable. From 
Tobias et al. (1954). 

the head of a rat. The hair of the animal turned white where the beam crossed 
the skin. It is impossible to tell which is the entrance or exit spot on the 
skin. There are many problems one encounters in actual practical radio- 
logic procedures. Accurate evaluation of the isodose cur\'es themselves pre- 
sent a great problem, since the dose varies very quickly with depth. The 
usual technicjue used is the exposure of a spectroscopical quality of photo- 
graphic material to the beam in an accurately constructed phantom with 
identical apertures and rotations as in the actual biologic exposure. One may 
then evaluate the dose distribution from photographic densitometry. Any 
such pi-ocedure presupposes knowledge of the dose-effect relationship in 
photographic film (Tochilin et al, 1955; Welch, 1955). Some corrections 
are necessary for various factors, e.g., oblique incidence of the beam, spread 
caused by motion of particles in air, etc. Other useful methods are solid 
state dosimeters, dosimetry by radioactivation, or the use of a chemical 
oxidation-reduction system. 

At the Berkeley 184 in. cyclotron, a de\ice is available for exact position- 
ing, by means of x-ray diagnostic techniques, of any desired part of the head. 
With the use of a three-dimensional Descartes coordinate system, it is pos- 
sible to deliver a radiation lesion of arbitrary size to the desired point. It is 
then possible, by merely changing one single coordinate, to deliver another 
lesion in the exact bilateral location. Usually the head of the animal that 



LESIONS WITH ACCELERATED PARTICLES 335 

is to be exposed is rotated around the point to be irradiated, while a cylin- 
drical beam constantly radiates the center of rotation. Use of the Bragg 
ionization peak for such irradiation is also possible, but more elaborate 
preparations are necessary, since the depth of the range needs to be con- 
trolled at all times. 

RaDIOBIOLOGIC ClONSIDERATIONS 

The nature of the radiation effect on the adult central nervous system 
poses great and as yet unsolved problems. The tissue has heterogenous archi- 
tectm~e: its structme is built for optimum maintenance of its most essential 
elements, the neurons, and this function is accomplished by an intricate 
architecture of the \aried cellular components. Proliferating elements of 
neural tissue are the astrocytes and ulial cells. Xeiuons in the adult do not 
seem to have mitotic acti\ity. and their functions appear to be mainly in 
transmission of action potentials and. in some instances, perhaps neurosecre- 
tion. Our cellular radiobiologic knowledge comes mainly from studies of 
rapidly proliferatino cells in the coinse of the cell division process, and we 
know that the maximum killing effect expresses itself mainly during and 
following the cell di\ision process. 

Since neurons do not divide, it is no wonder that they are thought to be 
relati\ely radioresistant. However, we do know that the developing embryo 
ner\ous system is extremely radiosensitixe see R. Ruyh. 1961 i. and 
studies in progress also point to radiosensitivity of the adult brain: usually, 
however, a considerable time elapses before the damage is developed to the 
point where it is pathologically obser\able. The observed change is usually 
necrosis, that is. disappearance of all cellular elements. Cmrent radiation 
studies point to the need of understanding the detailed biochemical and 
cellular processes of necrosis and its initiation. 

There is no definite dose-effect relationship as yet established for nerve 
tissue. We do know that therapeutic x-ray irradiation of a large part of the 
human brain may result in late degenerative changes, including demyeliniza- 
tion. scarring, and necrosis ( Druger ct al., 1954). Lindgren (1958) has 
collected human material and demonstrated that protracted irradiation 
schedules are less effecti\ e in causing necrosis than a single dose and that the 
dose-efTect relationship for protracted dose schedules is similar to that 
demonstrated by Strandquist (1944) for skin. Arnold et al. (1961) studied 
radiation effects on brain of primates and found demyelinization. the first 
observable chronic deleterious effect, one that may be caused by a dose of a 
few hundred rads. Lindgren's 19581 data on rabbits indicates somewhat 
higher dose thresholds. At Berkeley many irradiations have been carried out 
on animals oxer the last few years, usually with the proton or deuteron beam, 



336 



CORNELIUS A. TOBIAS 



in well-localized regions of the cortex, thalamus, brain stem, spinal cord, or 
cerebellum. Generally two relationships were observed: first, the higher the 
single dose, the sooner the onset of degenerative changes. Secondly, the 
larger the irradiated volume, the sooner the degenerative changes appear. 
Combining these two observations, we found it possible to compute the 
"integral dose,'' that is, dose times volume irradiated. As seen in Fig. 5, the 
integral dose and the time of death due to necrosis correlate reasonably well. 
Recently, Zeman ct al. (1959) produced radiation lesions in rats with very 
small beams of alpha particles. It was found that many tens of thosuands of 
rads are necessary to cause an eflfect as seen from Fig. 5. 

The effects of protracted dose schedules and of the size of the irradiated 
volume make it appear that some other factors enter in brain radiosensitivity 
besides direct interaction of radiation with neurons. 

One suspects the state of the capillary bed as being essential for the in- 
tegrity of neurons, particularly knowing that proper oxygen supply or nutri- 
tion is essential for the maintenance of the latter. Capillary walls are known 
to regenerate more efTectively following protracted dose schedules than a 
sino^le large dose. Certainly vasodilation following radiation and increased 
permeability of the blood-brain barrier have been observed in the early post- 
irradiation period (see communications by Van Dyke et al, Haymaker ct al., 
this conference.) 




Fig. 5. Relation.ship of integral dose to time of death due to necrosis and hemor- 
rhage in a number of experiments. 



LESIONS WITH ACCELERATED PARTICLES 337 

In the course of localized irradiation of the brain, special attention is 
given to the possibility that if the body and nucleus of neuronal cells are in 
the radiation field, the effect might be more profoimd than if only nerve 
trunks are irradiated. Unfortunately, so far there seems to be no definite 
evidence for such effect: in fact, demyelinization of fibers seems to occur 
chronologically earlier than some othe effects. However, it is possible that fol- 
lowing proper doses, fibers recover or even regrow from initial damage, while 
it is anticipated that neurons might not recover from nuclear damage. 

Early radiation-induced changes include alteration of the blood-brain 
barrier. If the lesion is massive, increased intracranial pressure might be one 
of the early gross signs of damage. It is possible that electric disturbances, 
resembling epilepsy, which sometimes follow massive head irradiation are 
in part due to the increased intracranial pressure. Tilsjar-Lentulis and 
Tobias ( 1959) in our laboratory observed cortical potentials following radia- 
tion in rats and foimd that the epileptic fits were absent when the brain was 
decompressed due to opening a large bone flap. In fact, using the cyclotron 
radiation technicjue it was foimd that irradiation of one cerebral hemisphere 
with doses in excess of 100,000 rads leads to immediate cessation of elec- 
trical activity in that hemisphere. The other irradiated hemisphere still had 
some acti\ity, but the alpha i hythm was about three times slower than 
normal. Changes in excitability and transmission as a result of local irradia- 
tion in various parts of the brain are the object of current studies. 

Stiiml'l.\tion by Means of Ionizing Particles 

The usefulness of nuclear beams in neurology would become even greater 
if one were able to use radiation to stimulate nerve discharge, perhaps in 
well-defined locations inside the brain. Several classes of observed psycho- 
logic effects of radiation (e.g., see Miller et ai, this conference) indicate 
that neuronal function is altered in some way following radiation, and we 
do know that the retina can be directly stimulated with low doses of x-rays 
(Lipetz, 1955). Hug (1960) has recently demonstrated interesting responses 
in snails. Conard (1957) followed radiation effects in muscles of rabbit 
intestine following radiation exposure. In preliminary studies at Berkeley, it 
was realized that instantaneous stimulation of action cinrents in nerve might 
recjuire high instantaneous radiation intensity for a brief period of time in 
a similar fashion as high electric current densities are required for brief 
periods of time to stimulate action currents. 

The Heavy Ion Linear Accelerator was chosen because it could deliver 
pulses of one millisecond duration, up to 10" rad/min in intensity. For the 
initial work a reflex was chosen which can be very easily demonstrated : the 
corneal blinking reflex. As seen in Fig. 6, the cornea of unanesthetized rabbits 



338 



CORNELIUS A. TOBIAS 



RECORDING EEG 



PRE-AMP 



HILAC. 
beam 




PRE-AMP 



H.V 



o 



^A Oscilloscope 




Beam focusing magnet 



Foil monitor Absorbers Rabbit (in holder) 



INTEGRATING 
DOSIMETER 



Fig. 6. Method of exposure of rabbit cornea to pulsed heavy ion beams. One or 
more radiation pulses of about 2 millisec duration are allowed to fall on the cornea. 
The beam pulse as well as contraction of the auricular muscle are simultaneously re- 
corded, and the blinking directly observed by closed circuit television. 



was exposed to one or more pulses of accelerated alpha particles, over an 
area 5 mm in diameter. The response of the auricular muscle was followed 
by recordino- the potential between the electrodes inserted into it. (Tobias 
et at., 1961). It was found that single or multiple pulses of beam did not 
cause blinkino- if less than about 20,000 rad was delivered within 1 sec to the 
substantia propria. Pidses larger than the threshold of about 30,000 rad per 
pulse did cause reflex muscle action with a delay of about 0.2-0.3 sec. By 
adjusting the range of the alpha particles it was also found that maximum 
stimulation occ tared when the Bragg ionization peak is at depth 100-200 fi 
under the corneal siuface; local anesthesia by a few drops of tetracaine 
abolished the effect. Such blinking reflex cotild occur if light, secondary to 
radiation, falls on the retina. That this was not a major part of the effect, 
was demonstrated by showing that the response is still present when the optic 
nerve is cut. The energy required for stimidation is sufficient to raise the 
local temperatiu'e (at the Bragg ionization peak) by about 0.1°C; it is 
known that absorption of infrared rays of about the same energy content 
can also initiate the blinking reflex (Dawson, 1961). Figure 7 reproduces 
some typical electrical records obtained in the course of radiation stimula- 
tion. The dose in a single beam pulse is sufficient to cause permanent patho- 
logic changes; these are presently being studied by S. Kimura. 

The corneal fibers that respond to such stimulation are believed to be pain 
fibers: it is possible that other type nerve endings or synapse will exhibit dif- 
ferent and perhaps greater radiation sensitivity. At the present time one 



LESIONS WITH ACCELERATED PARTICLES 



339 



Rabbit #2-9 




BEAM MONITOR 



1 2 3 4 5 6 

Seconds 

Fig. 7. Typical record of stimulation of blinking reflex. 

would not wish to use such large dose stimulation in humans. In animals 
this may be a different problem. We know that needles used in stimulation 
inflict permanent traiuna on brain tissue. It may well be that radiation- 
induced trauma will actually be less. 

Looking into the futme. one might \ isualize some of the advantages of 
hea\y ion pidses for stinudation and study of the cerebral cortex. One should 
probably try to stimulate with hea\ier ions than alpha particles; perhaps 
e\en a single accelerated hea\y nucleus of some ion such as iron might be 
capable of stimulating, owing to its sreat linear energy tiansfer. It seems 
quite possible to produce microbeams of heavy ions, perhaps 10 /x in diameter, 
then direct pulses of such beams to arbitrarily selected spots in the brain, and 
to vary the depth of penetration at will. One must admit that due to scatter- 
ing and straggling the resolution becomes poorer as the beam reaches to 
greater depth in the brain. Thinking about future possibilities for radiation 
stimulation of the brain. I wish to present a preliminary scheme of the Heavy 
Ion Scanning Stimulator i HISS ) . shown in Fig. 8. which is in construction 
at Berkeley. Here magnetic fields pro\ide a pre-coded scan pattern, as the 
beam is pulsed at some predetermined sequence. The response of the brain 
to such space and time coded radiation "messages'" to the surface of the 
cerebral cortex might prove to be illuminating to our knowledge of cerebral 
cortical function and behaxiour. 

Summary 



The chronological development of techniques for use of accelerated ions 
in neurologv are described. Methods for deep localization of lesions, produc- 
tion of laminar lesions, and knife-edge lesions are described. The stimulation 
of reflex action by alpha particles is demonstrated. 



340 



CORNELIUS A. TOBIAS 



Diagnostic x ray 




PROGRAMMER 



Fig. 8. Schematic concept of future uses of heavy ion pulses for conveying "coded" 
messages to the brain. The coding may occur by (a) magnetic deflection of beam in 
predetermined sequence, e.g., scanning; (b) by passage of beam through absorbers of 
predetermined profile to position the Bragg ionization peak where the stimulation 
occurs; (c) by coding time sequence and intensity of pulses of beam; (d) by using 
"feedback" information from periphery to change coded messages. 



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LESIONS WITH ACCELERATED PARTICLES 343 

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Effect of Local Irradiation of the Central 
Nervous System with High Energy Protons 

Bengt Axderssox. Borje Larsson, Lars Leksell. \Villiam Mair, 
Bror Rexed. axd Patrick Souraxder 

Gustaf Werner Institute for Nuclear Chemistry and 

the Institute of Anatomy, 

University of Uppsala, Sited en 



Hish ener^ry beams of licht atomic nuclei have physical properties which 
make them suitable when small volumes of tissue are to be unifomily irradi- 
ated (Tobias et al., 1952 i. Anderson et al. (1957) made lesions in the hypo- 
thalamus of the rat usins: 190 Mev deuterons. Malis ct al. (1957). making 
use of the thin ionization peak at the end of the beam, produced laminar 
lesions at a depth of 0.8 mm in the cerebral cortex of the cat with 10 Mev 
protons. Larsson et al. (1958) biiefly described several types of localized 
lesions in the central nervous system within 2 months after irradiation by a 
185 Mev proton beam. The histopatholooy of lesions produced by this tech- 
nicjue in the spinal cord and brain of the rabbit was described in detail by 
Larsson et al. (1959) and Rexed et al. (1960). Zeman et al. (1959), when 
investioatino- the effects of primaiy cosmic rays on nervous tissue, irradiated 
mouse brains with 22.5 Mc\- deuterons and studied the relationship between 
the size of the impact area and the threshold dose for a radiogenic lesion. 
Larsson i I960: described blood \essel changes following local irradiation of 
the rats brain with high energy protons. 

The present report is a summary of the neuropathologic findings in rab- 
bits and goats after local irradiation with a beam of 185 Mev protons from 
the 230 cm synchrocyclotron at the Gustaf Werner Institute, Uppsala. Lesions 
up to about 1 year are considered. The experiments were performed to get 
fundamental data concerning local effects of hea\y particle beams on the 
central nervous system. ha\'ing in mind their possible application to stereo- 
taxic neurosurgery. 

Material and Methods 

L-radiations were performed with a beam of 185 Mev protons from the 
svnchrocyclotron as described by Larsson et al. (1959). By focusinu' magnets 

345 



346 ANDERSSON, LARSSON, LEKSELL, MAIR, REXED AND SOURANDER 

and sweeping coils an almost parallel beam with a uniform cross section of 
variable size was obtained. The desired beam profile was determined by a 
final aperture. The mean dose-rate was 1,000-2,000 rad per min. The doses 
were measured with an ionization chamber and by activation dosimetry. The 
results obtained by the two methods were in fair agreement. Single proton 
beams were used for irradiation of the rabbit's spinal cord and brain. Cross 
fire irradiation was applied in the experiments on goats. 

In 55 rabbits and 5 goats, 3 types of lesions were produced : transection of 
the spinal cord, transverse lesion of the cerebral hemispheres, and restricted 
lesion in the depth of the brain. 

The spinal cord provides a suitable region to evaluate the effect of radia- 
tion on nervous tissues because of its regular arrangement of gray and white 
matter, and because disturbances of function are easy to observe. Proton 
beams, 1.5, and 10 mm broad, were directed across the spinal cord under 
deep intravenous nembutal anesthesia. The position of the animal was 
checked by roentgenograms before and after irradiation. After irradiation 
the animals were examined regularly, and neurologic symptoms noted. In 
most cases, paresis of varying degree developed. The animals were sacrificed 
by exsanguination under chloroform anesthesia at different periods following 
appearance of paresis. 

In a second series of experiments performed in rabbits, the 1 .5 mm proton 
beam was directed across the upper anterior part of the brain. A dose of 20 
krad was used, llie skull was fixed to a frame to avoid movement. During 
irradiation the animals were observed on a television screen. The animals 
were allowed to survive from 2 to 56 weeks. During this time no functional 
disability occurred which could be ascribed to the lesion. 

In view of the possible application of irradiation with high energy pro- 
tons to neurosurgery, experiments on goats were performed. The goat has a 
rigid skull which can be conveniently fixed in the same stereotaxic apparatus 
as that constructed for man. The goat's brain is rather large, so that the 
volume of necrotized tissue can be made relatively small compared to the 
size of the whole brain. This fact reduces the risk of side efTects, e.g., general- 
ized edema of the brain. One goat was irradiated with a single beam of pro- 
tons (dose = 20 krad). In 3 goats a disc-shaped lesion with a diameter of 
about 1 cm was produced in the internal capsule by the stereotaxic method 
using cross fire irradiation. A beam 2 mm x 7 mm or 2 mm x 10 mm and 
20-22 fields were used (the dose at the desired site was in 2 cases 20 krad 
and in 1 case 38 krad ) . The head of the goat was fixed to the stereotaxic in- 
strument by drills as described by Leksell (1951, 1957). When cross fire ir- 
radiation was applied, a point in the right internal capsule was selected as 
center of rotation. The lesions were planned so as to appear transverse to the 
direction of the fibers of the internal capsule. Before the operation, the goats 



LOCAL IRRADIATION OF CNS WITH HIGH ENERGY PROTONS 347 

were oiven chloralhydrate by a stomach tube, and later on, nembutal anes- 
thesia was inducted intravenously. The goats were killed 1 to 4 months after 
irradiation by decapitation under nembutal anesthesia. 

Spinal cords and brains were fixed in 5^? neutral formal-saline. From the 
irradiated part and adjacent regions of the cord, longitudinal and serial sec- 
tions were cut in a frontal plane. The rabbit brains were cut sagittally, and 
the slices were embedded in paraffin or cut in the frozen state. The goat 
brains were cut horizontally in slices about 5 mm thick and examined macro- 
scopically. The target region was embedded in celloidin and cut by serial 
sectioning. The principal staining methods used were hematoxylin and van 
Gieson's mixture, phosphotungstic acid hematoxylin, Heidenhain's modified 
Mallory stain for collagen, thionin. Grosthionin and Palmgren's silver impreg- 
nation for axons. Loyez and Heidenhain's myelin sheath stains, and Ranke's 
Victoria blue for astrocytes. In a few cases, supplementary stains for demon- 
stration of iron and fat were used. 

Results 

/' 

Transection of the Spinal CIord 

Eight rabbits were exposed to a single dose of 20 krad with a 1.5 mm beam. 
The first neiu'ologic symptoms, consisting of lively tendon reflexes in the 
hind legs and slight knee and ankle clonus, appeared about 8 days after irra- 
diation: later, flaccid paralysis developed. In the meninges and cord, a band 
of grayish discoloration 2 mm wide marked the radiated portion of the cord. 
No hemorrhages or inflammatory signs were visible to the naked eye. Micro- 
scopic examination revealed a narrow and sharply delimited necrotic zone 
corresponding to the path of the beam ( Fig. 1 ) . The lesion was broader in 
the white substance and had about the same breadth as the proton beam. 
The damaged gray matter was only about half the breadth of the beam 
(no correction for shrinkage due to fixation was made). In the path of the 
beam, nerve cells, axons, myelin sheaths, and glial cells were necrotized. No 
evidence of selecti\e radiation effect on the various components of the neural 
tissue was seen. Minimal hemorrhages surrounding capillaries and venules 
were observed at the margin and sometimes in the middle ot the irradiated 
zone. Large hemorrhages were never seen. Nuclear fragments, slight prolif- 
eration of astrocytes with bulky cytoplasm, and a few macrophages were 
noted in a small marginal zone between the irradiated and nonirradiated tis- 
sue (Fig. 2). Occasional nerve cells adjacent to the irradiated zone were 
degenerated, others being rather well preserved. In the white substance, the 
marginal zone was characterized by degenerating axons and myelin sheaths. 
Iron pigment occurred in a few glial cells at the edge of the necrosis. Fat 



348 ANDERSSON, LARSSON, LEKSELL, MAIR, REXED AND SOURANDER 




Fig. 1. Longitudinal section of rabbit's spinal rord; myelin jjicparation showing 
localized damage 12 days after irradiation with a 1.5 mm beam of high energy pro- 
tons. Dose: 20 krad. Heidenhain. X 9. The photographs are published by courtesy of 
Acta RadioJoQica. 



stain was always negative. The occurrence of a small number of well pre- 
served axons and myelin sheaths in the subpial region of the irradiated part 
of the cord was remarkable and constant. 

To study the relationship between the breadth of the beam and the type 
of radiation damage, a group of animals was irradiated with a beam 10 mm 
broad, the dose being the same as in the first series. Three to 4 days after 
irradiation, rapidly progressing paresis developed in the hind legs. These 
animals were killed 1 to 6 days after irradiation. Macroscopically, hyperemia 
of the meninges and spinal cord corresponding to the path of the beam was 
seen 1 to 3 days after irradiation, and numerous petechial bleedings 3 to 6 
days after irradiation. The earliest histologic changes foimd, at 1 and 2 days 
after irradiation, consisted of dilated capillaries and veins containing large 



LOCAL IRRADL\TION OF CXS WITH HIGH ENERGY PROTONS 349 






*i 


:.-5^- 


•i? ^ 




•,, 


, 


^ ^ • 






• 








* 

J* 


• • 


•• 






_ » *• 


*- • 




• . * 


/. « 




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^i r r 


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Fig. 2. The sharp demarcation of the irradiated zone in the gray matter of the 
spinal cord is seen to the right. Bordering the irradiated zone is a band of nuclear 
fragments and adjacent to them numerous astrocytes with' swollen cytoplasm and a 
few macrophages; further away rather well preserved nerve cells are observed. Proton 
beam 1.5 mm broad. Dose: 20 krad. Thionin. X 150. The photographs are pub- 
lished by courtesy of Acta Rddiologica. 

agcTi-eoations of erythrocytes. The ner\e cells, axons, myelin sheaths, and glial 
cells appeared to be intact. After 3 days the uray matter within a zone about 
10 mm wide showed exudation of plasma as well as multiple, and sometimes 
rather large, hemorrhages. The nerve cells were degenerating. The white 
matter was relatively spared and showed only slight perivascular disintegra- 
tion of the nervous tissue. Slight inflammatory cellular proliferation was noted 
in the meninges and cord corresponding to the irradiated region. At 4 to 6 
days after irradiation, widespread deueneration and necrosis of the white 
matter and abimdant hemorrhages in the gray columns were seen ; Fig. 3). 
The approximate relationship between dose and time of appearance of 
sisns was studied in a series ot animals irradiated with 5 to 40 krad. Animals 
irradiated with 12 krad or less did not, over a year, show any clinical signs 
or histologic changes in the cord. After a dose of 40 krad. paralysis of the 
hind legs developed in 2 days. The histologic picture 5 days after irradiation 
re\ealed necrosis considerably more extensi\e than that seen after a dose of 
20 krad. In addition, numerous small hemorrhages appeared. 



350 ANDERSSON, LARSSON, LEKSELL, MAIR, REXED AND SOURANDER 








Fig. 3. Myelin preparation showing extensive necrosis and multiple perivascular 
hemorrhages 4 days after irradiation with a 10 mm high energy proton beam. Dose: 
20 krad. Hcidcnhain. X 9. The photographs are published by courtesy of Acta 
Radiologica. 



Transverse Lesion of the Cerebral Hemispheres 

Twenty rabbits were irradiated with a 1 .5 mm broad beam directed trans- 
versely through the upper part of the frontal lobes. The dose was 20 krad. 
The rabbits remained functionally unaffected during the time they were 
allowed to survive, up to about 1 year. Macroscopic changes consisted of a 
red or green groove on the anterosuperior surface of the brain, and in some 
instances, on the medial surfaces of the hemispheres as well. No large hemor- 
rhage was seen, and the leptomeningeal vessels in the groove were not throm- 
bosed. Sagittal sections of the brain showed a well demarcated lesion in the 
cortex, white matter, and caudate nucleus, which up to 3 months after irra- 
diation was approximately restricted to the path of the beam. After 
3 months the lesion was slightly broader than the beam (Fig. 4). 



LOCAL IRRADL\TIO\ OF CNS WITH HIGH ENERGY PROTONS 351 












• •■. ^i. . ■ ./ ■ ',• 
•^:•■•;■.■ •, ' '■■:^i'S'.. 



s 



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S6,-\ .Vo >-sN\' 

^ .-..^-X.*^: > i- ■'■S*i ff"«k... jV-?.. > «- ^^ 



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Fig. 4. Sagittal mx liuu of rabbit's brain 13 wcrks after irradiation with a 1.5 mm 
beam of high energy protons. A discrete lesion corresponding to the path of the beam 
is seen. The cortical laminae are interrupted and the myelin destroyed. Increased 
vascularity is noted in the irradiated region. Dose 20 krad. Loyez stain for myelin. 
X 45. llie photographs are published by courtesy of Acta Radiologica. 



352 ANDERSSON, LARSSON, LEKSELL, MAIR, REXED AND SOURANDER 

Two weeks after irradiation, collections of fluid and small perivascular 
hemorrhages were observed in the irradiated zone. Within the lesion many 
thick-walled capillaries lined by large endothelial cells were present. The 
nerve cells, axons, and myelin sheaths in the path of the beam were mostly 
destroyed. In the damaged tissue, proliferation of astrocytes had occurred, 
and collections of macrophages were seen, particularly around the vessels. 
Four weeks after irradiation, cells with large nuclei, 2 or 3 times the nor- 
mal size, were seen among the proliferating astrocytes. Numerous large atypi- 
cal glial cells appeared within 3 months after irradiation (Fig. 5). Some of 







Fig. 5. Atypical glial cells appeared in the lesion at 4 weeks after irradiation and 
were more frequent at 10 weeks. Thionin. X 640. The photographs are published by 
courtesy of Acta Radiologica. 



these were binucleated or multinucleated; many had a huge nucleus of 
bizarre shape. Inclusion bodies were frequently seen in the nuclei of these 
cells. Some of the giant cells resembled nerve cells in that they had a large 
nucleus with a distinct nuclear membrane and a central nucleolus; the nu- 
cleus was often eccentric, and there was a large amount of glassy cytoplasm. 
The various types of atypical cells were most frecjuent at the margins of the 
lesion. They were never seen in other parts of the brain. 




LOCAL IRRADIATION OF CNS WITH HIGH ENERGY PROTONS 353 

Cavities of varying size and shape were inacroscopically seen in about 
half of the irradiated rabbits. They involved mainly the white matter, but 
in some cases the cortex was also affected (Fig. 6). Cavitation was first ob- 
ser\ed 10 weeks after irradiation. In some animals, surviving up to a year, 
no cavities appeared. The cavities were not encapsulated, but were lined by 
glial cells and giant cells and contained clear ffuid and macrophages. 

Teleangiectasis of the irradiated region was a striking feature. It 
was present in all animals surviving more than 23 weeks. The cortex and 
white matter exhibited numerous blood-filled, thin-walled capillaries of wide 
caliber lined by a single layer of endothelial cells. Occasionally some of these 
capillaries were thrombosed, and sometimes small hemorrhages and collec- 
tions of hemosiderin-filled phagocytes were found around them (Fig. 7). 
Large hemorrhages were never encountered. 

A thin zone of the cortex immediately underlying the pia matter showed 
less damage than the rest of the irradiated cortex. 

It was an unexpected finding that the ependyma and choroid plexus al- 





'V, 



V 



V 



Fig. 6. Sixteen weeks after irradiation. The damaged region shows loss of substance 
and the formation of small cavities in the deeper parts of the cortex and white matter. 
Ependyma undamaged. The lesion is slightly broader than the beam. H. \an G. X 20. 
The photographs are published by courtesy of Acta Radiologica. 



354 ANDERSSON, LARSSON, LEKSELL. MAIR. REXED AND SOURANDER 




> u - t^J^v * ** :> X ?* '. 




Fig. 7. Twenty-three weeks after irradiation. Newly formed wide vessels lined with 
flattened endothelium and surrounded by macrophages, some of then containing hemo- 
siderin, now appear in the lesion. H. van G. X 200. The photographs are published 
by courtesy of Acta Radiologica. 

ways were unafTected. though the proton beam often passed through parts 
of these structures. 

Restricted Lesion in the Depth of the Brain 

To be able to compare the radiogenic brain lesions in rabbit and goat with 
each other, 1 goat was irradiated with a single beam directed through the 
frontal lobe of the right cerebral hemisphere (dose =^ 20 krad). The brain 
was examined 1 month after irradiation. The lesion in this goat bore a 
strong resemblance to the lesions in the brains of rabbits irradiated under 
similar conditions. 

Lesions were produced in the internal capsule of 3 goats by the stereotaxic 
method using cross fire irradiation. A dose of 20 krad was given to 2 of these 
goats, 'iliey remained in good condition and did not show any symptoms of 
cerebral injury. Four and 7 weeks after irradiation, they presented restricted 
lesions in the selected areas (Fig. 8). The damaged region was roughly lenti- 
forni, with a maximum thickness in the anterioposterior direction of about 
3 mm. The longest diameter of the lesion was in the dorsoventral direction 



LOCAL IRRADL\TION OF CXS WITH HIGH ENERGY PROTONS 355 




Fig. 8. Horizontal section of the right hemisjjhere of a goat's brain showing a 
sharply deHmited lesion in the internal capsule 7 weeks after cross-fire irradiation with 
20 krad. Gros-thionin. X 5. The photographs are published by courtesy of Acta 
Radiolosica. 



and measured 13-16 mm. 7 he histopathologic picture of the brain lesions of 
these 2 coats corresponded closely to each other. Within the white matter the 
lesion was sharply limited, and its central part was completely necrotic. The 
capillaries were destroyed, and aroimd them were seen shadows of erythro- 
cytes and plasma exudate. There were no lar^e hemorrhaues inside the lesion 
or around it. A narrow zone smroundino- the definite lesion showed desjener- 
atino axons and myelin sheaths. Slight cellular proliferation was noted. In the 
lesion examined 7 weeks after irradiation, a few macrophages were seen 
around distended vessels at the margin of the damaged resion. Outside the 
lesion there were no signs of pathologic processes in the brain. 

A dose of about 40 krad given by cross fire irradiation to 1 goat produced 
a different lesion. The volume of necrotic tissue was large. The tissue around 
the necrotic area in some places contained wide, thin-walled, newly fonned 
vessels and was densely infiltrated with lymphocytes and macrophages. The 
affected region surroimdino- the necrosis was at least the size of the necrotic 
area itself. Onlv minimal hemorrhages were noted in the outer zone. The 
irradiated hemisphere was swollen, but did not show any other pathological 
chano;es outside the lesion. 



356 ANDERSSON, LARSSON, LEKSELL, MAIR, REXED AND SOURANDER 
Discussion 

The proton beam as an agent for production of lesions in the central 
nervous system has the advantage that the particles of the beam proceed 
along practically straight tracks and undergo little scattering. The ionizing 
effect is thereby restricted, and it should be theoretically possible, therefore, 
to cause a sharply defined lesion. 

The lesions in nervous tissue caused by high energy protons v^as that of 
an acute necrosis and similar to that described by Arnold and co-workers 
(1954) after irradiation of the brain of the monkey with high energy roent- 
gen radiation. In our experiments on the spinal cord, as well as in those by 
Arnold and co-workers (1954) on the brain of the monkey and by Larsson 
(1960) on the brain of the rat, it appeared that the latent period for radi- 
ation necrosis depends on the dose of radiation. A dose of 20 krad with a 
1.5 mm-broad proton beam gave a well defined lesion after 9 days. With 
larger and smaller doses, the latent period was shortened or prolonged, 
respectively. A radical increase of the dose above 20 krad, considered opti- 
mum dose, induced in the spinal cord, as well as in the brain, a more 
widespread tissue reaction. This observation stresses the importance of select- 
ing the dose carefully when planning any surgical procedure. 

The breadth of the beam proved to be a factor of great importance. In 
the experiments on the spinal cord a 10 mm beam not only produced 
necrosis of about the same breadth as the beam, but also produced a lesion 
considerably more quickly and of a histologically more severe type than a 
thinner 1.5 mm beam. With the thin beam, only slight vascular damage 
and minimal hemorrhages were seen. The same dose and a broad beam 
caused, even after 1 day, signs of vascular damage, and after 3 days, plasma 
exudation and numerous hemorrhages, especially in the gray substance. 
It is assumed that the peculiar type of \ascular arrangement in the spinal 
cord and the poor development of collaterals in its thoracic part favored 
the appearance of large hemorrhages when a considerably large portion of 
the cord was irradiated. 

In experiments on the tolerance of mouse brain tissue to high energy 
deuterons, Zeman and co-workers (1959) fomid that the dose required to 
produce a threshold lesion in mouse brain increased from 30 krad with a 
beam 1 mm broad to 1.1 X 10'' rad with a beam 0.025 mm in diameter. 
According to these authors, a possible explanation would be that the micro- 
beams cause a predominantly direct radiation injury, whereas the broad 
beams produce additional indirect effects, e.g., vascular disturbances. 
Arnold and co-workers using 10 or 25-mm-broad beams of high energy 
roentgen rays did not find evidence that the lesions produced by broad 
beams were secondary to vascular occlusion. In our experiments the narrow- 



LOCAL IRRADL\TIO\ OF CNS WITH HIGH ENERGY PROTONS 357 

est beam used was 1.5 mm broad, and lesions produced by this beam were 
not studied before 9 days after irradiation. When the 10 mm-broad beam 
was used and the histolooy of the spinal cord studied 1 to 6 days after 
irradiation, vascular changes were found at the time when only slisjht 
destruction oi myelin sheaths had appeared. This obser\ation stresses the 
possible importance of \ascular changes amon^" the causal factors of delayed 
radionecrosis in the nervous system. Furthermore, the presence of a thin 
peripheral zone ol the cord with undamaoed ner\e fibers may speak in 
favor of vascular factors, since it is well known that the peripheral region 
of the cord is relati\ely resistant to impaired circulation. The studies of 
Larsson 1960) on blood \essel chanscs following local irradiation of the 
rat's brain with high energy protons indicate that disturbances in capillary 
circulation may precede increased permeability of the blood-brain barrier 
system for trypan blue. The latter type of changes was accompanied or 
succeeded by destruction of nervous tissue. 

In the rabbit's brain irradiated with high energy protons, capillary pro- 
liferation and sometimes teleangiectasis appeared within the lesion. It might 
be supposed that such changes of vessels would predipose to hemorrhage, 
but in fact a large hemorrhage was never seen either in the late or early 
stages after irradiation. Teleangiectasis was also obser\"cd by Berg and 
Lindgren i 1958 I in the rabbit's brain after roentgen irradiation. 

The various types of giant cells found at the margins of the radiolesion in 
rabbit's brain were probably of glial origin and resembled the cells foimd 
in gliomata. These cells were always confined to the margins of the lesion 
and were ne\er found to have spread to other parts of the brain. The 
ninnber of the giant cells did not increase during the experiment of one 
year. Exceptionally large astrocytes were described in rabbits after roentgen 
irradiation of the brain by Russell ct al. : 1949 ) . and giant, atypical glial cells 
ha\c been obserxed in primates by Arnold and Bailey 1954 i and Berg and 
Lindgren il958). 

The experiments reported show that, with a narrow beam of high energy 
protons, sharply delimited acute lesions at a desired site in any region in the 
central nervous system can be produced. Furthermore, it is obvious that by 
careful selection of doses and dose distributions, well circumscribed intra- 
cerebral lesions of appropriate size and shape can be caused. However, this 
statement is valid only for the earlier stages of the radiolesion, as only 
relatively short survi\al periods are considered. To what extent high energy 
protons may be used in stereotaxic surgery of the hiunan brain depends on 
the late changes. The long-term effects of high energy proton irradiation 
are at present being studied in a series of experiments on goats, in which 
deep lesions are to be followed up over several years. 



358 ANDERSSON, LARSSON, LEKSELL, MAIR, REXED AND SOURANDER 

Summary 

For the past several years experiments were carried out to determine the 
local efTect on the central nervous system of his,h energy protons. The Irra- 
diations were performed on rabbits and goats with a 185 Mev proton beam 
from the 230 cm synchrocyclotron at Uppsala. The experiments have shown 
that It Is possible with high energy protons to produce small well circum- 
scribed cerebral lesions at a desired site in the brain without damaging the 
surrounding tissue. The Importance of performing long-term experiments to 
determine the late effects of high energy protons on the brain is stressed. 

References 

Anderson, A., Garcia, J., Henry, J., Riggs, C, Roberts, J. E., Thorell, B., and Tobias, 

C. A. 1957. Pituitary and hypothalamic lesions produced by high-energy deutcrons 

and protons. Radiation Research 7, 299. 
Arnold, A., and Bailey, P. 1954. Alterations in the gHal cells following irradiation of 

the brain in primates. A.M. A. Pathol. 57, 383-391. 
-Arnold, A., Bailey, P., and Laughlin, J. S. 1954. Effects of betatron radiations on the 

brain of primates. Neurology 4, 165-178. 
Berg, N. O., and Lindgren, M. 1958. Time-dose relationship and morphology of de- 
layed radiation lesions of the brain in rabbits. Acta Radiol. Suppl. 167, 1-118. 
Larsson, B. 1960. Blood vessel changes following local irradiation of the brain with 

high-energy protons. Acta Soc. Med. U psaliensis 65, 61-71. 
Larsson, B., Leksell, L., Rexed, B., Sourander, P., Mair, W., and Andersson, B. 1958. 

The high-energy proton beam as a neurosurgical tool. Nature 182, 1222-1223. 
Larsson, B., Leksell, L., Rexed, B., and Sourander, P. 1959. Effect of high energy 

protons on the sj)inal cord. Acta Radiol. 51, 52-64. 
Leksell, L. 1951. The stereotaxic method and radiosurgery of the brain. Acta Chir. 

Scand. 102, 316-319. 
Leksell, L. 1957. Gczielte Hirnoperationen. In "flandbuch der Neurochirurgie" (H. 

Olivecrona and W. Tonnis, eds.). Vol. VI, pp. 178-199. Springer, Berlin. 
Leksell, L., Larrson, B., Andersson, B., Rexed, B., Sourander, P., and Mair, W. 1960. 

Restricted radio-lesions in the depth of the brain produced by a beam of high 

energy protons. Acta Radiol. 54, 251-264. 
Malis, L. J., Loevinger, R., Kruger, L., and Rose, J. E. 1957. Production of laminar 

lesions in the cerebral cortex by heavy ionizing particles. Science 126, 302-303. 
Rexed, B., Mair, W., Sourander, P., Larsson, B., and Leksell, L. 1960. Effect of high 

energy protons on the brain of the rabbit. Acta Radiol., 53, 289-299. 
Russell, D. S., Wilson, C. W., and Tansley, K. 1949. Experimental radionecrosis of 

the brain in rabbits. /. Neurol. Neurosurg. Psychiat. 12, 187-195. 
Tobias, C. A., Anger, H. O., and Lawrence, J. H. 1952. Radiological use of high 

energy deuterons and alpha particles. Aryi. J. Roentgenol. Radium Therapy Nuclear 

Med. 67, 1-27. 
Zeman, W., Curtis, H. J., Gebhard, E. L., and Haymaker, W. 1959. Tolerance of 

mouse-brain tissue to high energy deuterons. Science 130, 1760-1761. 



Production of Laminar Lesions in the 
Cerebral Cortex by Deuteron Irradiation 

Leonard I. Malis. Jf:rzv E. RosE.f Lawrence Krugkr.} 

AND C^HARLES P. BaKER 

Mt. Sinai Hospital, Xeiv York, New York, 

Johns Hopkins University School of Medicine. Baltimore, Maryland, and 

Brookhaien National Laboratory, Upton. Xezc York 



Our interest in radiation has been aroused by the possibiHty of iitilizins: the 
cyclotron beam for investigation of connections and functions of the cerebral 
cortex. 

The mammalian cerebral cortex consists of an outer layer, almost devoid 
of nerve cells, and of several inner layers, more or less densely packed with 
radially oriented ner\e cells. This makes it appear laminated. The functional 
sionificance of this lamination is obscure. We thought it of interest to pro- 
duce lesions restricted to one or several layers in order to study them 
electrophysiologicallv and histoloeicallv. 

Other workers employing hea\y. ionizing particles usually used a high 
energy beam. With such a beam, one can irradiate with only that portion 
of the beam path along which the loss of energy is almost uniform. The 
terminal .sector of the beam path i with great difTerences in energy loss) is 
arranged to lie outside the irradiated organism. With this technique, it is 
possible to produce substantially uniform destruction along the beam path if 
the dose is high enough. Destruction deep within the brain can be achieved 
by multiple irradiations. The beam path for each irradiation traverses dif- 
ferent sectors of the brain, and the intensity of each is below threshold lor 
destruction of the tissue. All beam paths, however, are made to con\erge on 
the target area, which thus recei\es the desired radiation dose i Anderson et 
al, 1957a.b; Larsson ct ai, 1959: Tobias ct ai, 1954). 

In contrast to these techniques introduced by Tobias and his co-workers 
(1954 k we ha\'e used a beam of only moderate energy which is stopped 
entirely within the cerebral cortex. With such a beam, it is fairly simple to 

* Supported in part by a research grant from the National Institutes of Heahh and 
by the U.S. Atomic Energy Commission. 

t Present address: Uni\-ersity of Wisconsin. Madison. Wisconsin. 

+ Present address: L'ni\ersity of Ciahfornia Medical Center. Los Angeles. California. 

359 



360 



MALIS, ROSE, KRUGER AND BAKER 



produce a restricted lesion deep in the cortex with a single irradiation, since 
one can take advantage of a sharp increase in the number of ionizations pro- 
duced for a short distance near the end of the beam path, just before all 
ionizations drop abruptly to zero as the particles are stopped by the tissue 
(Malis t'^ al., 1957, 1958). 

Figure 1 illustrates a typical laminar lesion 65 days after irradiation with 
20 Mev deuterons. The laminar lesion is a sharply demarcated strip com- 
pletely free of nerve cells. There is a noticeable loss of nerve cells in cortical 
layer IV just above the laminar lesion; the still more superficial cortical lay- 
ers (II and III) and the cortical zonal lamina (layer I) appear substantially 
intact. 

A laminar lesion covers an area commensurate with the aperture used. Its 
dorsoventral dimensions are always limited, though the width of the lesion is 
a function of the radiation dose. With smallest effective doses, the width of 




Fig. 1. Laminar lesion (1) in the striate (St) and peristriate (Ps) areas of a rabbit. 
The extreme edge of the lesion just extends into the posterior limbic region (PI). 
Due to the scatter of the beam, the lesion is narrower in the peristriate than in the 
striate field. 65 days after irradiation. Peak dose: 33,000 rads; average dose: 12,000 
rads; surface dose: 7,000 rads; number of deuterons per cm": 8.8 X 10" I=lst 
cortical layer, II — IV=cellular cortical layers. X 30. 



LAMINAR LESIONS IN CEREBRAL CORTEX 



361 



the lesion may be as narrow as 60 a*: with higher doses which produce 
typical laminar destructions this width is close to 180 m; still higher doses 
may produce lesions over 200 M wide. (All the figures refer to stained 
preparations. ) 

One striking observation in regard to the laminar lesions is that their 
glial content is moderate in animals sur\iving 6 weeks or longer. In Fig. 1 
the glial content of the laminar lesions appears approximately the same as 
that of the zonal lamina. This is typical, even though a laminar lesion in 
early stages displays a vascular and glial reaction, which may be intense 
if the radiation dose is nearly maximal for laminar destruction. 

We have conclusive evidence that older laminar lesions regularly display 
dendritic processes and a wealth of axons which form irregular and often 
denser patterns than do normal cortical sectors (Figs. 2-4). In Figs. 2-4, 
showing a laminar lesion in the upper part of layer VI 204 days after irradi- 
ation, it is apparent that the laminar lesion is filled with ner\'e fibers that 
form a dense, abnormal striation, which rather taithtully lollows the undula- 
tions of the laminar lesion. Fronr a study ot a large amoimt of material (pre- 




FiG. 2. Laminar lesion (1) in the postcentral region of another rabbit 204 days 
after irradiation. .Almost normal cortical sector at the extreme left of the figure. Peak 
dose: 27,000 rads : a\'erage dose: 9,000 rads: surface dose: 6,000 rads: number of 
deuterons per cm': 7.4 X 10''. Formalin fixation, frozen section, Nissl stain, X 50. 



362 



MALIS, ROSE, KRUGER AND BAKER 




Fig. 3. Section adjacent to that shown in figure 2. Schultze's silver stain for fibers. 
Laminar lesion (1) is filled with nerve fibers which form a dense striation and inter- 
rupt the orderly radial arrangment of the fiber bundles. Notice the near normal appear- 
ance of the remaining portion of the cortical field. X 60. 



pared with a \ariety of histologic techniques i from brains of animals surviv- 
ing from several days to 18 months after irradiation, we concluded that many 
nerve fibers in the laminar lesion must be new sprouts. 

We have reported on the technique as well as on the histologic appear- 
ance of the laminar lesions and the problem of fiber growth in considerable 
detail elsewhere (Malis ct al., 1960; Rose ct al., 1960). However, consider- 
ing the interest in the problem of the radiation dose, we will repeat a few 
points concerning the technique as well as some problems arising in regard to 
the determination of the dose. 

All irradiations were carried out with the beam from the 60 in. cyclotron 
at Brookhaven National Laboratory, which delivers 20 Mev deuterons, 10 
Mev protons, or 40 Mev alpha particles. Irradiations were done mostly with 
the deuteron beam, because its range in the brain (about 2.5 mm) is twice 
as large as the ranges of available protons or alpha particles. Figure 5 illus- 
trates the irradiation arrangement. The evacuated beam pipe brings the col- 



LAMINAR LESIONS IN CEREBRAL CORTEX 



363 




Fig. 4. Area enclosed by the rectangle in Fig. 3 shown imder magnification of 150. 



liniated, '■detocused" beam from the cyclotron. Measurements ot the current 
in an aroon-filled, transverse ionization chamber, placed at the end of the 
beam pipe, were made to calculate the radiation doses. After the beam left 
the ionization chamber, it traxersed usually 334 in. of air before strikint;' the 
taroet. A rifle telescopic sisht with an illumination de\"ice and a mirror was 
mounted on a slide on the ionization chamber, so it could be moved with 
precision into alignment, and the taroet area centered in the cross hairs of 
the telescope. With suitable calibrations, the telescope served to center the 
target in relation to the beam and to bring the target to a predetermined 
distance from the ion chamber. 

The aseptic operation consisted of removal of the bone, preparation of 
a bloodless field, and closure after irradiation. All irradiations were done 
with dura intact. 

Figure 6 shows the well known Bragg curve. The curve, for 20 Mev deu- 
terons. plots the relative ionizations after penetration of aluminum foil 
against the thickness of the foil. There is a sharp rise in number of ions 
produced just before the end of the range, and the value for the ionization 
peak is nearly 5 times higher than at the beginning of the range. This sharp 



364 



MALIS, ROSE, KRUGER AND BAKER 




Fig. 5. Scheme of the irradiation arrangement. The telescope, which indicates the 
position of the target area in relation to the beam and the distance of the target from 
the ion chamber, is shown in the position it occupies during irradiaton. An absorber 
(often used to shorten the range of particles in tissue) is advanced in the absorber 
rack to a distance of about 12 mm from the target to minimize the effects of scatter. 
The absorber does not touch the target in order not to compromise the sterility of 
the exposed cortex. The animal is positioned in relation to the beam in the milling 
machine. 



rise in ionization near the end of the rans,e makes possible the production of 
a laminar lesion. Considering that one mil thick aluminum foil is nearly 
ecjuivalent in its stopping power to 50 fx of brain, one can estimate the prob- 
able width of laminar destruction from the ionization curve. There is a 
reasonable, thotigh not strict, relationship between the expected and meas- 
ured thicknesses of the laminar lesions for light and moderate radiation 
doses. Since the zone of maximal ionization occurs near the end of the beam 
path, it is readily possible to shift this zone, and hence the locus of the lami- 
nar lesion, if the range of particles is shortened in tissue by introducing 
energy-degrading materials into the beam path. It follows, from considera- 
tion of the stopping power of brain and aluminum, that introduction of one 
mil thick aluminiun foil elevates the zone of maximal ionization in the brain 
by about 50 /x. 

Several difficulties arise in the determination of the radiation dose. The 
first group of problems pertains to the determination of the actual number 
of particles which strike the target in a given irradiation arrangement. 
Further difficulties occiu' when one wishes to express the radiation dose in 
a manner which has biologic meaning, once the number of particles bom- 
barding the target is known with reasonable accuracy. 



LAMINAR LESIONS IN CEREBRAL CORTEX 



365 




MILS ALUMINUM 5 
189 177 



4 8 ENERGY OF 

DEUTERON IN MEV 



Fig. 6. Bragg ionization curve. Relative ionization produced by tiie beam of deuter- 
ons in a thin ionization chamber after passage through ahmiinum foil of varying 
thickness. The actual energy of the particles, as delivered by the cylcotron, was slightly 
higher than 20 Mcv. Since, however, some absorbers had to be introduced into the 
beam path to make the determinations shown, the first value for relative ionizations 
is given for deutcrons of 18.9 Me\- energy. Ordinate: amount of ionization current in 
arbitrary units per constant nimiber of incident deuterons. Abscissa: upper row of 
figures: thickness of aluminum foil in mils; lower row of figures: the remaining energy 
of deuterons in Mev after passage through aluminum foil of stated thickness. 



The first oroiip of problems is teclmical. We have used for calibration 
purposes measurements of radioacti\ity induced by irradiations of copper 
and lead discs. It was necessary to introduce correction factors for the non- 
linearity of the ion chamber and for the scatter of the beam. 

Assuming," that our calibrations are reasonably satisfactory, it is possible to 
express each irradiation dose as the nianhcr of diutcrons per cm-. Since 
knowlcdoe of the number of deuterons per cm- is essential for expressing the 
dose in any other way, it is apparent that this number is a straightforward 
measurement of the radiation dose. From a biologic point of view, the dose 
so expressed permits a reasonable estimate of the number of ions per imit 
area which may be relatively harmless, injurious, or lethal for a neuron or 
nerve fiber. Oiu' data suggest that mild to hea\y laminar lesions are produced 
by doses of 4 x 10" to 12 x 10'' deuterons per cm'-, respectively. 

The basic disad\antage in expressing the dose as a number of deuterons 



366 MALIS, ROSE, KRUGER AND BAKER 

per cm- is made clear by the consideration (see Fig. 6) that the number of 
ions produced per unit length of path varies for different sectors of the path 
and that it is the number of ions produced per unit length of path and not 
the number of particles which traverse the cortex which is responsible for 
the laminar destructions. 

One does not avoid this essential disadvantage by computing the dose in 
rads, if the computation is made on the basis of number of particles, their 
energies, and their range in tissue. The figure which emerges from such cal- 
culation represents an average dose in rads, which would be an adequate 
measure only if the energy loss along the beam path were uniform. Since 
this is not the case, it is desirable to estimate the actual doses delivered at 
chosen points along the beam path. We have routinely computed such doses 
at two levels. The first is the dose in rads delivered to the surface of the 
cortex {surface dose) ; the second is the dose delivered at the ionization peak 
{peak dose). For calculation of the surface dose, the rate of energy loss must 
be known. For brain, this rate was considered equal to the appropriate values 
tabulated for water (Rich and Madey, 1954), since the rates of energy loss 
for brain and water cannot be significantly different. Once the surface dose 
is determined, the peak dose (or any other dose at a desired depth) can be 
estimated from the ionization curve (Fig. 6), if a reasonable assumption is 
made that the shapes of the ionization ciuves in brain and aluminum do not 
diflfer materially. 

The peak dose is, we believe, the most appropriate measure of the radia- 
tion effect in our material, despite the fact that its estimate is technically 
less reliable than that of other measures. The peak dose is the highest dose 
delivered over a short distance in tissue. It can be argued from the ionization 
curve in Fig. 6 that values close to the peak dose can be expected to be 
delivered to a strip of cortex about 50 p. wide. 

The peak dose, the surface dose, and the a\erage dose have constant rela- 
tionships to each other if the range of particles in tissue remains the same. 
If the range is shortened in tissue by introduction of an absorber into the 
beam path, the ratios change, since the value of the peak dose remains con- 
stant, while the average dose and the surface dose become higher. In our 
irradiation arrangements, the peak dose was usually almost 5 times larger 
than the surface dose and about 3 times larger than the average dose. 

As is known, some time elapses before a radiation lesion manifests itself 
histologically. The latent periods tend to be long with marginal doses and 
become greatly reduced as the dose increases (Malis et al., 1960). We will 
consider the dose necessary to produce a laminar lesion in rabbits which 
survived from 3 weeks to 18 months after irradiation. Table I assembles such 
data for 177 lesions. Each lesion was studied in serial sections cut at 30 fx, 
and every section was mounted. 



LAMINAR LESIONS IN CEREBRAL CORTEX 367 

TABLE I 

Relation' of Peak Doses to Histological Fixdinos 

Number and type of lesions " 

Peak doses '' No evidence 

(in 1 ,000 rods) of lesion Light laminar Heavy laminar Necroti foci 

< 14 6 9 — — 

15-24 1 16 — — 

25-34 _ 20 10 2 

35-44 _ 8 25 16 

45-54 _ _ 7 29 

>55 -- — 4 24 

•' Findings for 177 radiation lesions in rabbits survi\ing from 2l! to J48 days. 
'' Siiigle irradiations with 20 Mcv deuterons. 

The lesion is defined as litiht laminar if the destruction is restricted to the 
zone of maximal ionization throughout the entire irradiated region. It is 
called a heavy laminar lesion if the band of destruction is broader and some 
changes are manifest for a short distance above the zone of maximal ioniza- 
tion. A lesion is classified as showing a necrotic focus if anywhere within the 
irradiated region there is a sign of vascular occlusion or its sequelae. Only 
some lesions in the last category (produced by very large doses) display signs 
of radiation necrosis throughout the entire irradiated region. 

The table implies that each type of lesion can be produced by a large 
span of radiation doses, even if one assumes that some extreme figures in 
each category merely represent an apparent spread due to errors in determi- 
nation of the deli\ered doses. Several conclusions are apparent. A peak dose 
of about 30,000 rads (which corresponds in our irradiation to a surface dose 
of approximately 6,000 rads and an a\ erage dose of about 10,000 rads) leads 
almost invariably to excellent laminar destruction. A peak dose up to 45,000 
rads often produces hea\y laminar destruction; on the other hand, a peak 
dose can be reduced to about 15,000 rads and still almost always result in 
a narrow, light laminar lesion. Peak doses below 15,000 rads may fail to 
produce lesions, at least for many weeks or months, while doses in excess of 
45.000 rads can be expected to produce necrotic foci. 

It appears that a peak dose of about 15,000 rads can be tentatively 
accepted as a reasonable approximation of a minimal dose, which must be 
applied over a short distance in the cortex to produce total destruction of 
nerve cells within a few weeks. It follows from this consideration that a 
surface dose of about 15.000 rads (which roughly corresponds to a peak dose 
of 75.000 rads) should be necessary to produce total necrosis of the irradi- 
ated cortex with our beam. This expectation seems in reasonable agreement 



368 MALIS, ROSE, KRUGER AND BAKER 

with our observations, since no peak dose in our material smaller than 65,000 
rads produced total necrosis. 

References 

Anderson, A., Garcia, J., Henry, J., Riggs, C, Roberts, J. E., Thorell, B., and Tobias, 
C. A. 1957a. Pituitary and hypothalamic lesions produced by high-energy deuterons 
and protons. Radiation Research 7, 299. 

Anderson, A., Roberts, J. E., Thorell, B., and Tobias, C. A. 1957b. Arrest of growth 
in young rats after hypothalamic deutcron irradiation. Radiation Research 7, 300. 

Larsson, B., Leksell, L., Rexed, B., and Sourandcr, P. 1959. Effect of high energy 
protons on the spinal cord. Acta Radiol. 51, 52-64. 

Malis, L. I., Locvinger, R., Kruger, L., and Rose, J. E. 1957. Production of laminar 
lesions in the cerebral cortex by heavy, ionizing particles. Science 126, 302-303. 

Malis, L. I., Kruger, L., and Rose, J. E. 1958. The use of deuterons in production of 
laminar lesions in the cerebral cortex of the rabbit. Trans. Am. Neurol. Assoc. 
pp. 78-80. 

Malis, L. I., Baker, C. P., Kruger, L., and Rose, J. E. 1960. Effects of heavy, ionizing, 
monoenergetic particles on the cerebral cortex. I. Production of laminar lesions and 
dosimetric considerations. /. Comp. Neurol. 115, 212-242. 

Rich, M., and Madey, R. 1954. Range-Energy Tables, Univ. of California Radiation 
Lab. Document UCRL-2301 1-433. 

Rose, J. E., Malis, L. I., Kruger, L., and Baker, C. P. 1960. Effects of heavy, ionizing, 
monoenergetic particles on the cerebral cortex. II. Histological appearance of 
laminar lesions and growth of nerve fibers after laminar destructions. /. Comp. 
Neurol. 115, 243-296. 

Tobias, G. A.. Van Dyke, D. D., Simpson, M. E., .\nger, H. O., Huff, R. L., and 
Koneff, A. A. 1954. Irradiation of the pituitary of the rat with high energy deu- 
terons. Am. J. Roentgenol., Radium Therapy Nuclear Med. 72, 1-21. 



Fluorescein as a Sensitive, Semiquantitative 
Indicator of Injury Following Alpha Particle 
Irradiation of the Brain* 

D. C. Van Dyke, P. Janssen, and C. A. Tobias 

Donner Laboratory, University of California, Berkeley, California, and 
the Armed Forces Institute of Pathology, Washington, D.C. 



Soluble fluorescein U.S. P. i Uranine I . a dye routinely used by physicians 
as a convenient and sensitise indicator of damati,e to corneal epithelium, has 
been found to be an equally con\enient. semiciuantitative, and sensitise indi- 
cator of irradiation injury to brain tissue. The use of this method as an indi- 
cator ot dama(;e to brain tissue is based on the same piinciple as its use as an 
indicator of corneal epithelial damage, i.e., normal brain tissue, like normal 
corneal tissue, does not stain, whereas injured tissue of both types readily 
takes up the dye. The method is Cjuantitative, since the intensity of staining 
is dependent on the sexerity and number of injured cells. Permeability of 
the tissue to fluorescein represents a physiologic or biochemical alteration 
of the cell which precedes morphologic changes demonstrable by light 
microscopy. 

The inxasion of brain tissue by cells which are normally permeable to 
fluorescein can be recognized by the fact that they fluoresce following admin- 
istration of the dye. This fact has been successfully used as a technicjue to 
locate brain timior tissue at surgery (Moore, 1953). 

This paper describes the use of fluorescein staining as a sensitive and semi- 
quantitati\e indicator of injury to brain tissue following localized irradiation 
with a beam of alpha particles from the 184 in. cyclotron. The method has 
been used to demonstrate the difference in radiosensiti\itv of brain tissue in 
difTerent species. 

Material and Methods 

Male rats of the Long-E\ans strain, 28 clays of age at the time of irradia- 
tion, were used, except when the eflect of age difference was in\estigated. 
Young adult cynomolgus monkeys and rabbits of the New Zealand White 

* Supported in part by the U.S. .-Xtomic Energy Commission. 

369 



370 



D. C. VAN DYKE, P. JANSSEN AND C. A. TOBIAS 



strain were used in the comparison of the radiosensitivity of various species. 
During irradiation all animals were given light barbiturate anesthesia, and 
their heads were firmly mounted in specially designed holders (Fig. 1 ) . Rigid 
fixation of the heads of all animals was accomplished by the use of ear plugs 
and a sharp pin into the gingiva and maxilla between the dorsal incisors. To 
keep the level of barbiturate anesthesia light, it was necessary to use xylo- 
caine anesthesia locally in the ear canals and gingiva of the monkeys and 
rabbits. 

Proper anatomic positioning of the beam was insured by having an x-ray 
souixe aligned with the path of the beam, so that a roentgenogram of the 




Fig. 1. During irradiation all animals were given light barbiturate anesthesia, and 
their heads were firmly mounted in a specially designed holder. For proper positioning 
of the beam, a "Land" camera was placed in the path of the beam beside the animal's 
head. 



skull with the image of the alpha particle beam superimposed could be 
made for each animal. For this purpose a "Land" camera was placed in the 
path of the beam beside the animal's head (Fig. 1). Figure 2 is such a 
"Land" camera roentgenogram of a rat's skull with the image of the 2.0 X 
25.5 mm beam superimposed. Such pictures served as a permanent record 
for later comparison with the position of the band of fluorescence or the 
position of miscroscopic changes. 

A 2.0 X 25.5 mm brass aperture 6 in. thick was used, except in the experi- 
ment where the effect of aperture size was specifically studied. The distance 



FLUORESCEIN AS INDICATOR OF BRAIN INJURY 371 




Fig. 2. A "Land" camera roentgenogram of a rat's head with tlie image of the 2.0 
X 25.5 mm beam superimposed. 

between the aperture and the area of the brain to be studied was kept con- 
stant ( L75 in.) . 

Fkiorescein stainin^ of the damaged brain tissue was accompHshed by 
intravenous administration of fluorescein sodium ( 100 mg per ml, pH 8.2) 
under ether anesthesia of 10 mo- per 60 2,m body weight. After 45 minutes 
the animal was anesthetized with ether, and the head was removed by Guillo- 
tine and immediately frozen in liquid nitrogen. The head was then sectioned 
on a high-speed handsaw in the sagittal plane, which was 1.75 in. from the 
aperture at the time of irradiation. The cut sin-face was polished by holding 
it under rimning water until free of all dust fragments, but not long enough 
to melt the tissue. The two halves were examined in a dark room imder 
uItra\iolet light while solidly frozen. A \isual estimation of the intensity of 
the fluorescent band was recorded. Evaluation of the fluorescein staining 
was made on the basis of an arbitrary scale from 1^- to 4+, with 1-|- being 
faintly \isible and 4-|- being intense. When subsequent histologic examina- 
tion was desired, the frozen brain was immersed directly in formalin and 
processed in the usual way. Rapid freezing with liquid nitrogen did not 
seriously interfere with later microscopic evaluation of the tissue. Micro- 
scopic examination was also made of brain tissue which had not been sub- 
jected to freezing to insure accurate interpretation. The skull vvas carefully 



372 D. C. VAN DYKE, P. JANSSEN AND C. A. TOBIAS 

opened and the head immersed in 10'"r formalin. The brain was removed 
from the skull the followins: day and placed in fresh formalin. When 
thoroughly fixed, the irradiated area was blocked out, dehydrated in graded 
alcohols, and embedded in paraffin. Sections were cut at 6 ^u, and routinely 
stained with hematoxylin-eosin, \an Gieson, Kr thionin solution for Nissl 
substance, and Palmgren's silver stain for fibers. 

Throughout this study, 900 Mev alpha particles from the 184 in. cyclotron 
were used. All effects described resulted from a single dose given at a dose 
rate of 2,500 rad per min. 

The beam was monitored with a parallel plate ionization chamber with 
aluminum foil electrodes and nitrogen atmosphere (Tobias et al, 1952; 
Birge, ct al, 1956). For absolute standardization, this ion chamber was cali- 
brated against a Faraday cage, which was used to measure the particle ffux 
in the beam. The distribution of beam intensity after the beam passed through 
the aperture was calibrated with photographic emulsion dosimetry. By this 
technicjue, dose of beam measured by the monitor ion chamber was allowed 
to pass through the aperture and fall on a photographic film exposed in a 
phantom to correspond with the position of the irradiated animal's head. 
Densimetry of the photographic film yielded information for dose distribu- 
tion. Dose values are in rads and refer to peak dose at the central plane of 
the knife-edge lesion. 

Results 

The minimum dose which would produce changes and the time of onset 
of demonstrable radiation changes in the brains of rats were studied by 
giving single doses from 5,000 to 26,000 rads and autopsying the rats at 
postirradiation intervals from 6 hours to 100 days. The results were evaluated 
by the presence of fluorescein staining and by morphologic changes seen with 
light microscopy. Figure 3 summarizes the results obtained in rats, using 
fluorescein staining as the criterion of damage. The time of onset and inten- 
sity of fluorescein staining is different for diff"erent doses of irradiation. At 
the lowest doses of 5,000 and 6,000 rad, no staining occurs until several 
weeks after irradiation, the intensity of staining is low, and the eflfect appears 
to be transient. Following doses of 26,000 rad, there was definite staining 24 
hours after irradiation and liquifaction of the irradiated zone by the 3rd 
postirradiation day. Doses of irradiation differing by 20'"r betw^een these two 
extremes can be clearly separated both as to time of onset and intensity of 
staining. Figure 4 is a log-log plot of the time of first appearance of fluo- 
rescence as a function of dose and serves to illustrate the definite relationship 
between dose and time of onset of recognizable changes (staining with 
fluorescein) . 



FLUORESCEIN AS INDICATOR OF BRAIN INJURY 



373 



S?+++ 




12 16 20 24 28 32 36 40 

Days after irradiation 
Fig. 3. Summaiy of results obtained using fluorescein staining as a criterion of 
damage in rats. The time of onset and intensity of fluorescein staining are different for 
diflferent doses of irradiaton. 



1 St a ppearance of 

histologic lesior 



•- — 



1st appearance of fluorescenc 



01 1 10 100 

Days after irradiation 

Fig. 4. .\ log-log plot of the time of first appearance of fluorescence as a function 
of dose, illustrating the definite relationship between dose and time of onset of recog- 
nizable changes (staining with fluorescein). 



Microscopic examination of the same brains that were examined for fluo- 
rescence, as well as of brains from companion rats from whom the tissue 
was removed and fixed without pre\ious freezino, demonstrated that a 
lesion is seen earlier and at lower doses with fluorescein staining than with 
light microscopy. Alter a dose of 8.000 rad. a fluorescein-stained lesion was 
clearly demonstrated during the 2nd and 3rd weeks postirradiation without 
subsequent clearly demonstrable microscopic e\idence of damage. 



374 D. C. VAN DYKE, P. JANSSEN AND C. A. TOBIAS 

After a dose of 10,000 rad, no microscopic lesion was recognizable dur- 
ing the first few weeks (none by 37 days) , but eventually (84 days or more) 
a minimum lesion appeared in the irradiated area. 

At doses above 10,000 rad, a microscopic lesion appeared earlier and at 
a dose of 20,000 eventually (6 weeks postirradiation) led to complete necro- 
sis and removal of the irradiated zone with healing of the margins. At 20,000 
rad, the first recognizable morphologic changes were seen by the 5th day. 

Because doses of irradiation differing by 20 9r can easily be difTerentiated 
by the fluorescein technique, it was thought that this method might be useful 
in demonstrating whether such variables as age of animal, size of aperture, 
or type of particle would affect the result. 

A dose of 10,000 rad appeared to be ideal for demonstrating any differ- 
ences in effectiveness, because at this dose there is a definite time lag fol- 
lowed by a plateau in response which clearly differentiates it from the next 
highest or next lowest dose (12,000 or 8.000 rad). 

The effect of age of the rat on flourescein staining of the irradiated brain 
was studied using 22-day- and 120-day-old rats, a dose of 10,000 rad, and a 
postirradiation interval of 7 days (4 rats of each age) . The results in the two 
groups were identical, 3 rats of each group being classed as l-j- and 1 from 
each group being classed as 2-|-, a response which could not be expected 
from a dose effect equivalent to 8,000 or 12,000 rad. Thus, the appearance 
of the fluorescent lesion is not affected by the age of the animal, within the 
range studied. 

To determine the effect of size of lesion on flourescein staining of the 
irradiated rat brain, a study was made 7 days after irradiation with 10,000 
and 8,000 rad through a 4, 2, or 0.5 mm. slit aperture ( 2 mm used in all 
other studies). The exact dosimetry for the different apertures was deter- 
mined in the manner described. When the animals were autopsied, all brains 
given the larger dose showed a clear band of fluorescence corresponding to 
the position of the beam, and no brain given the smaller dose showed a 
lesion. Thus the dose-effect relationship appears to be independent of the 
size of the irradiated area within the limits studied, i.e., 0.5 to 4 mm. 

Two experiments were done to compare the effectiveness of a given dose 
of alpha particle irradiation in diflerent species. Rats, rabbits, and monkeys 
were given a single dose of 11,000 rad and examined at various postirradia- 
tion intervals. In the first series, the earliest time interval was 48 hours, by 
which time the monkey brain showed a 4-\- fluorescein staining and begin- 
ning licjuefaction of the irradiated area. The results from examination of 
fluorescein staining in the second series, in which the time intervals were 
shortened to get early observations on monkeys, are compared in Fig. 5. 
Five monkeys given 3,000 rad and autopsied at 3, 5, 8. 12, and 15 days 
showed no staining with fluorescein. By comparing the results from Fig. 5 



FLUORESCEIN AS INDICATOR OF BRAIN INJURY 



375 




2 4 6 S iC i2 14 

Days after irradiation 

Fig. 5. A comparison of the development of a fluorescent lesion in the brains of 
rats, rabbits, and monkeys, all gi\en the same dose (10,000 rad) through the same 
aperture on the same day. 



with the dose-response curve in Fio. 3. one sees that the response obtained 
in the rats in this experiment corresponded to a dose of approximately 1 1 ,000 
rad, whereas the response in rabbits corresponded to approximately 15,000 
rad, and the response in monkeys corresponded to something greater than 
26.000 rad. Fiaure 6A illustrates the intensity i4-k) of fluorescein staining 
of the monkey brain 24 hours after a dose of 11,000 rad. The photograph 
was taken while the brain was still frozen, using ultra\iolet ligiit. Rats gi\en 
the same radiation dose showed no fluorescein staining at 24 hoius and showed 
onlv 1+ fluorescence at 5 days. The monkeys killed at 24 and 48 hoias had 
generalized paralysis and periodic clonic con\ulsions, illustrating that not 
only their permeability to fluorescein but also the functional capacity of the 
tissue was altered. Figure 7 compares the morphologic changes seen in the 
monkey brain 24 hours after a dose of 11,000 rad with the morphology of 
the rat brain 24 days after the same dose delivered under identical conditions. 
Figme 7A is a low power photomicrograph of the irradiated portion of 
the brain from a companion monkey to that shown in Fig. 6A. The animal 



Fig. 6A. An illustration of the intensity (4 +) of fluorescein staining of the mon- 
key brain 24 hours after 10.000 rad. The photograph was taken while the head was 
still frozen, using ultraviolet light. Rats given the same radiation dose and killed at 
the same time showed no fluorescence. 

Fig. 6B."Land"' camera picture taken at the time of irradiation, superimposed on a 
roentgenogram of a monkey's skull for orientation. 



■■ 


^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^l^B 


^^^H 


^1 


^^^^^m< -.. ' ' '\ -,j9HHh ''^-"9 


^HRil 


1 






1 






■ 




^^H 


a| 




^^^H 




376 




'*«W«S9^- 



Fig. 7 a. A low power (H. & E. X 6.5) photomicrograph of the irradiated portion 
of the brain from a companion monkey to that shown in Fig. 6A. The animal was 
killed 24 hours after irradiation with 11,000 rad. The width and position of the beam 
is indicated. There was a 6-mm wide band of intense (4+) fluorescence extending 
34 of the way to the bottom of the section in the fresh specimen. The photograph 
illustrates the isolated patches of lightly stained tissue seen in the gray matter and the 
mottled appearance of the white matter in the path of the beam. 

Fig. 7B. Lightly staining patches in the gray matter enlarged 24 times. By 48 hours 
the entire irradiated area had become liquescent. 

377 



A^m,77- 





2mm 




I'lG. 7C. Rat brain (H. & E. X 6.8) 24 days after the same duse and aperture as 
used in the monkeys in Figs. 7A and 7B. Without magnification or with the help of 
low magnification, it can be seen that in the cortex there is a decrease in the staining 
quality of the ground substance with some capillary dilatation. No changes were 
recognized in the white matter (corpus callosum). 

Fig. 7D. Higher power magnification (X 26) of the area of the cortex showing 
changes. Microscopic examination of the brains of rats from the same group autopsied 
at 20 days or less after irradiation showed no recognizable morphologic changes. 



378 



FLUORESCEIN AS INDICATOR OF BRAIN INJURY 379 

was killed at 24 hours after irradiation with 11.000 rad. There was a 6 mm 
wide band of intense !4-|-) fluorescence extending 34 of the way to the bot- 
tom of the section in the fresh specimen. The photograph illustrates the 
isolated patches of lightly stained tissue seen in the gray matter and the 
mottled appearance of the white matter in the path of the beam. Figure 7B 
shows the lightly staining patches in the gray matter enlarged 32 times. By 
48 hours the entire irradiated area had become liquescent. 

Figure 7C shows a rat brain 24 days after the same dose and aperture as 
used in the monkeys in Fig. 7A and 7B. Without magnification, or with the 
help of low magnification, it can be seen that in the cortex there is a de- 
crease in staining quality of the ground substance with some capillaiy 
dilatation. No changes were recognized in the corpus callosum. Figure 7D is 
a higher power magnification of the area of the cortex showing changes. 
Microscopic examination of the brains of rats from the same group autopsied 
at 20 days or less after irradiation showed no recognizable morphologic 
changes. By 44 days after irradiation, the irradiated area had undergone 
hemorrhagic necrosis with a lethal subarachnoid hemorrhage occurring in 
some animals, a degree of pathology grossly comparable to that found in the 
monkey after 48 hours. A detailed study of the histopathology of such lesions 
has been presented by Janssen et al. '1961). 

Two methods of more accurately quantitating the extent of injury, using 
fluorescein as an indicator and as a method of evaluating the acciuacy of 
the arbitrary \isual grading of the lesions. ha\e been suggested. Photograph- 
ing the lesion, using standardized conditions under ultraviolet light, as in 
Fig. 6. and quantitating the film, using a micro-densitometer. could be done. 
Pieces of tissue of uniform size taken from the center of the lesion ha\e been 
analyzed for fluorescein content using a fluorospectrophotometer in a few 
cases. The values obtained for tissue which had been graded 1+, 2+, and 
4+ were 62. 78. and 460 jjsz fluorescein. 

Discussion 

The use of fluorescein staining as a criterion of brain injury following 
irradiation has been described in detail because it combines the advantages 
of being cjuick. simple. semiquantitati\e, and remarkably sensitixe for brain, 
rather than because of any basic physiologic or biochemical significance of 
the property of staining per se. The simplest explanation for the fact that 
the tissue takes up the dye onlv after injury would seem to be that the mem- 
branes of injured cells became permeable to substances which they would 
normally exclude. However, when considering brain tissue, one cannot ignore 
the so-called blood-brain barrier phenomenon which has been the subject of 
so much controversv i Clemente and Richardson. 1961 ) . This study does not 



380 D. C. VAN DYKE, P. JANSSEN AND C. A. TOBIAS 

provide evidence as to the mechanism involved in the acquisition of stain- 
abihty of brain tissue following" irradiation. 

It should be emphasized that the results presented in this paper both for 
fluorescein staining' and morphologic changes represent acute changes follow- 
ing high doses of irradiation and are for the most part confined to observa- 
tions during a 3 week postirradiation period. The possibility of profound 
changes occurring in the central nervous system many months or years after 
relatively low doses of irradiation has been adequately emphasized by other 
studies (Arnold ct ai, 1954c; Lindgren, 1958) and has not been considered 
in this work. 

The doses of alpha particle irradiation which must be given in order to 
produce changes in the central nervous system have been found to be rela- 
tively high (above 5,000 rad ) . Parallel studies done by Janssen et al. (1961) 
using alpha particle irradiation confirm the finding that a dose in excess of 
5,000 rad (6,000 was the minimum effective dose found by these authors) 
must be given before morphologic changes are observed at any postirradia- 
tion inteival in the rat. However, Hicks and Montgomery (1952) have shown 
that 6 to 24 hours after x-ray doses of as little as 1,200 rad to the head of 
rats, necrotic oligodendroglia cells were found scattered through the white 
and gray matter. 

The greater radiosensitivity of the primate brain as compared to that of 
the rodent brain which was found in this study both by the fluorescein 
staining technique and gross or microscopic morphologic examination, has 
been observed and emphasized by Arnold ct al. ( 1954a,b,c) and by Lindgren 
(1958) . The work of these authors indicates that the greater radiosensitivity 
of the primate brain occurs not only in the early postirradiation period after 
high doses, as was the case in this study, but also after long postirradiation 
periods and relatively low doses. Lindgren (1958) has emphasized that the 
radiosensitivity of the brain appears to vary from one region to another, 
cortex and the medullary region immediately underneath it being less radio- 
sensitive than deep-seated parts of the white matter ( Markiewicz, 1935; 
Pennybacker and Russell, 1948; Zeman, 1950). The radiosensitivity of the 
white matter also appears to vary from region to region, as is suggested by 
the disseminated appearance of the lesions on uniform irradiation of the 
entire brain (Scholz and Hsu, 1938) and by lesions seen in the midbrain 
(Arnold vt ai, 1954b). One must be cautious in generalizing about brain 
tissue as a whole, and cautious in comparing work done with diflferent types 
of radiation, different species, and different postirradiation intervals, until 
more data have accumulated. 

Monkeys given 11,000 rad showed a response greater than that loimd in 
rats after 26,000 rad, suggesting that at this dose the brain tissue of the 
monkey is almost 3 times more radiosensitive than the brain tissue of the 



FLUORESCEIN AS INDICATOR OF BRAIN INJURY 381 

rat. However, monkeys s,iven 3.000 rad showed no staining with fluorescein, 
suooesting that the dose response ctirve for monkeys woidd have a steeper 
slope than that for tlie rat when plotted as in Fia. 4. but that the monkey 
may not show a "greater radiosensitivity of brain tissue at lower radiation 
doses. The work of Hager ct al. (1961 ) using x-ray indicates that the ham- 
ster brain is probably cjuite radiosensitive. 

In an attempt to rule out the possibility that the data collected in this 
study pertained to the age of animal and aperture size, age and size of 
aperture were investigated. Rats 22 days old showed the same response as 
did rats 120 days old. There was no demonstrable difference in biologic 
efTecti\eness with apertiae sizes ranging from 0.5 to 4 mm. These studies 
tend to emphasize the species diflferences found and broaden their interpreta- 
tion, but are not intended to imply that a^e and size of lesion are not im- 
portant factors in more extreme situations. Hicks 1953) has demonstrated 
that the adidt ner\ous system is radioresistant and the embryonic nervous 
system radiosensiti\e, and Lindgren (1958) has stated that in clinical prac- 
tice it has pro\ed ad\isable to reduce the adult dose by 25''r for treating 
brain lesions in 5-year-old children and by 50''r when treating 2-year-old 
children. Zeman ct al. \ 1959) have demonstrated that in order to produce 
a lesion in the brain when the aperture is narrowed 25 p.) . an extremely 
high dose must be delixered to the tissue. Hicks ct al. < 1958) has demon- 
strated differences in radiosensitivity between strains of mice and difTerences 
in pathologic and physiologic effects when the intensity of the radiation is 
varied (Hicks ct al., 1956) . 



Summary 

Soluble fluorescein U.S. P. ( Uranine ) , a dye routinely used by physicians 
as a convenient and sensitive indicator of damage to corneal epitheliimi. has 
been found to be an ecjually con\enient. semiquantitative, and sensiti\"e indi- 
cator of injiny to brain tissue following localized irradiation with a beam of 
alpha particles from the 184 in. cyclotron. Permeability of the tissue to 
fluorescein represents a physiologic or biochemical alteration of the cell, 
which occurs before any morphologic changes demonstrable by light mi- 
croscopy. The time of onset and intensity of fluorescein staining are different 
for different doses of irradiation. 7 he minimimi effecti\e dose was 5.000 or 
6.000 rad. A species difference in radiosensitivity of brain tissue was demon- 
strated both by the fluorescein technicjue and by histologic morphology. The 
brain of rabbits and monkeys is considerably more sensitive to alpha particle 
irradiation than is that of rats. 



382 D. C. VAN DYKE, P. JANSSEN AND C. A. TOBIAS 

References 

Arnold, A., Bailey, P., and Harvey, R. A. 1954a. Intolerance of the primate brainstem 

and hypothalamus to conventional and high energy radiations. Neurology 4, 575- 

585. 
Arnold, A., Bailey, P., Harvey, R. A., Hass, L. L., and Laughlin, J. S. 1954b. Changes 

in the central nervous system following irradiation with 23-mev x-rays from the 

betatron. Radiology 62, 37-44. 
Arnold, A., Bailey, P., and Laughlin, J. S. 1954c. Effects of betatron radiations on 

the brain of primates. Neurology 4, 165-178. 
Birge, A. C, Anger, H. O., and Tobias, C. A. 1956. "Radiation Dosimetry" (G. J. 

Hine and G. L. Brown, eds.), pp. 624-662. Academic Press, New York. 
Clemente, C. D., and Richardson, H. E. 1961. Some observations on the effects of 

ionizing irradiations on blood vessels and the blood brain barrier. Symposium on 

the Response of the Nervous System to Ionizing Radiation, 1960, this volume. 

Chapter 25. 
Hager, H., Hirschberger. W., and Breit, A. 1961. Electron microscopic observations on 

The x-irradiated central nervous system of the Syrian hamster. Symposium on the 

Response of the Nervous System to Ionizing Radiation, 1960, this volume, Chapter 

16. 
Hicks, S. P. 1953. Effects of ionizing radiation on the adult and embryonic nervous 

system. Metabolic and toxic diseases of the nervous system. Research Pubis. Assoc. 

Research Nervous Mental Disease 32, 439-462. 
Hicks, S. P., and Montgomery, P. O'B. 1952. Effects of acute radiation on the adult 

mammalian central nervous system. Proc. Soc. Exptl. Biol. Med. 80, 15-18. 
Hicks, S. P., Wright, K. A., and Leigh, K. E. 1956. Time-intensity factors in radia- 
tion response. A.M. A. Arch. Pathol. 61, 226-238. 
Hicks, S. P., Wright, K. A., and D'Amato, C. J. 1958. Time-intensity factors in radia- 
tion response. A.M. A. Arch. Pathol. 66, 394-402. 
Janssen, P., Tobias, C. A., and Haymaker, W. 1961. Pathologic changes in the 

brain from exposure to alpha particles from a 60-inch cyclotron. Symposium on 

the Response of the Nervous System to Ionizing Radiation, 1960, this volume, 

Chapter 24. 
Lindgren, M. 1958. On tolerance of brain tissue and sensitivity of brain tumours to 

irradiation. Acta Radiol. Suppl. 170. 
Markiewicz, T. 1935. Uber Spatschadigungen des menschlichen Gehirns durch 

Rontgenstrahlen. Z. ges. Neurol, u. Psychiat. 152, 548. 
Moore, G. E. 1953. "Diagnosis and Localization of Brain Tumors." Charles C Thomas, 

Springfield, Illinois. 
Pennybacker, J., and Russell, D. S. 1948. Necrosis of brain due to radiation therapy. 

/. Neurol., Neurosurg. Psychiat. 11, 183. 
Scholz, W., and Hsii, Y. K. 1938. Late damage from roentgen irradiation of the 

human brain. Arch. Neurol. Psychiat. 40, 928-936. 
Tobias, C. A., Anger, H. O., and Lawrence, J. H. 1952. Radiological use of high 

energy deuterons and alpha particles. Am. J. Roentgenol., Radium Therapy Nuclear 

Med. 67, 1-27. 
Zeman, W. 1950. Die Toleranzdosis des Hirngewebes bei der Rontgentiefenbestrah- 

lung. Strahlentherapie 81, 549. 
Zeman, W., Curtis, H. J., Gebhard. E. L., and Haymaker, W. 1959. Tolerance of 

mouse brain tissue to high-energy deuterons. Science 130, 1760. 



Pathologic Changes in the Brain from 
Exposure to Alpha Particles from a 60 Inch 

Cyclotron * 

Peter Janssen, *■* 

Dormer Laboratory, Unirersity of California, Berkeley, California, 
Armed Forces Institute of Pathology, ]\'ashington, D.C. 

Igor Klatzo, Jaime Miquel, 

National Institute of Neurological Diseases and Blindness, 
National Institutes of Health. Bethesda, Maryland 

Tor Brustad, 

Norwegian Radium Hospital, Oslo, Norway 

Albert Behar, Webb Haymaker,*** 

Armed Forces Institute of Pathology. Washington, D.C. 

John L^man. Jilian Henry, and CIornelius Tobias 

Donner Laboratory, University of California, Berkeley, California 

Introduction 

This paper deals chiefly with time-dose relationships in the appearance 
of alterations in the cerebellum and cerebrum of rats followin" exposure to 
alpha particle radiation from the 60 in. cyclotron in Berkeley. Exposure 
of brains to protons was also done for comparison. We were concerned with 
( 1 ) severity of the lesions in terms of energy given off along the course of 
the Bragg cin\e, (2) alterations in structural elements with respect to time- 
dose relationships, and ( 3 ) circulatory and vascular alterations, including 
permeability changes as assessed by the Pickworth-Lepehne stain for erythro- 
cytes and by certain fluorescence indicators, i.e., fluorescein-labeled scrimi 
proteins (FLSP) and sodium fluorescein. 

Methods 

In earlier phases of the study with about 25 ''r of the animals, the top of 
the skull was removed (in a 15 x 10 mm area) and the dura left intact 

* This sUidy was supported by funds from the U.S. Atomic Energy Commission and 
the National Aeronautics and Space .Administration. 

** Present .Address: Institut Neurologique Beige, Brussels. Belgium. 

*** Present .Address: N.AS.A .Ames Research Center. MoflFett Field. California. 

383 



384 JANSSEN ET AL. 

(Simpson et al., 1953), but this practice was discontinued when it was found 
that adequate lesions could be produced through the intact skull. There- 
after the operation consisted simply of incision and reflection of the scalp 
prior to irradiation. The cyclotron aperture was circular and 14.3 mm in 
diameter, sufficient in size to allow irradiation of most of the dorsal surface 
of the cerebellum and cerebrum bilaterally. 

For morphologic studies, metallic impregnations for glia and Pickworth- 
Lepehne benzidine staining for visualization of the vascular tree were used. 
Usually the brains were fixed in Bouin's solution. Vascular permeability to 
serum proteins was studied by the FLSP technique (Klatzo and Miquel, 
1960). In this procedure, the rats were injected intravenously with 2 cc of 
8% fluorescein isothiocyanate-labeled albumin, usually 24 hours prior to 
sacrifice, and unstained, formalin-fixed, frozen sections were examined under 
the fluorescence microscope. For observations on vascular permeability to 
sodium fluorescein, animals irradiated at the same dose level were injected 
with 1 cc of 10% sodium fluorescein intravenously, usually 24 hours prior 
to sacrifice, and the gross observations for fluorescence were conducted as 
previously described (Klatzo ct al., 1958). 

The alpha particles and protons to which the dorsal part of the brain was 
exposed had an energy of approximately 12 Mev per nucleon. These par- 
ticles were first deflected away from the magnet and down the deflector 
channel, which contained a ^ mil Al stripping foil and a slit system to ex- 
clude particles of unsuitable energy or charge-to-mass ratio. To obtain a 
homogeneous particle flux distribution over the target area, a defocused 
setting of a pair of cjuadropole magnets was used. The beam then passed 
through a monitor, called a "high vacuum ionization chamber" (Brustad et 
al., 1960) , and finally out of the vacuum system through a 1 mil Al window. A 
specially constructed parallel plate ionization chamber (Brustad et al., 1960) 
was attached to the snout, and the animals to be irradiated were placed in a 
special holder about 5 mm from the end window of the ionization chamber. 
This air gap reduced the effective particle range in tissue by less than 5 /x. 
The ionization chamber could be detached and replaced by a magnetically 
guarded Faraday cup. The monitor upstream was always calibrated against 
the Faraday cup in terms of nimiber of bombarding particles per cm-. When 
animals were exposed, the ionization chamber response and the calibrated 
monitor response were recorded independently and converted to surface dose 
in rad. These two difTerent dosimeters generally agreed to within a few per 
cent. 

Between the ionization chamber and the monitor, sets of calibrated Al- 
absorbers could be introduced with a remote controlled absorber changer. 
With the Faraday cup connected, accmate determination of particle range 
and beam-energy homogeneity could be performed routinely. With the 



PATHOLOGIC CHANGES IN BRAIN CAUSED BY ALPHA PARTICLES 385 

ionization chamber connected, Brao-^ cui\es could be measured and con- 
verted to tissue equi\alent \alues. Depth-dose distribution in tissue was 
inferred from the measured surface dose by use of a Bragg cur\e derived in 
this manner (see Fig. 6). It was found that the effective range in tissue of 
the full energy protons and alpha particles (after allowance was made for 
the various absorbers, Al foils, and air gaps, which are permanently in the 
beam during exposure) was about 118 mg per cm-, or 1.180 /x, if a tissue 
density of 1 is assumed. Homogeneity of the particle flux over the target 
area was checked from densitometer readings of exposed films or from "burn 
patterns" obtained on exposed ozalid paper. When the flux distribution was 
unsatisfactory, adjustments were made with the focusing magnets and, if 
necessary, in the entire alignment of the experimental setup. Details of 
dosimetric procedure and of particle properties are described elsewhere 
(BrUstad ct al, 1960; Birge et al, 1956). 

The dose rate used dining the exposure of the rats was approximately 
10,000 rad per minute surface close, which corresponded to about 4 x 10'' 
particles per cm- per sec. Due to difficulties in the accelerator operation, a 
constant dose rate was sometimes difficult to maintain: fluctuations o\er a 
factor of 2 or more occurred. 

Observations 

The brain-surface doses of alpha particles and protons and the times of 
sacrifice of the animals are gi\en in Table L The brains were exposed to 
alpha particles at surface doses of 50 to 6,000 rad and to protons at a sur- 
face dose of 6,000 rad. 

Whether produced by alpha particles or by protons, the basic lesion tol- 
lowino irradiation consisted initially of a zone of cell damatic which stretched 
horizontally across the cerebellar and cerebral cortex. The lower border of 
the zone was sharp: the upper border tended to be indistinct. Within the 
deepest ])art of the zone of damage a ner\e-cell-poor or ner\e-cell-free hand 
eventually appeared. The width of the zone and of the band \aried with 
the radiation dosage. In the text which follows, the terms "'zone"" and ■"band" 
are used in the sense indicated. 

Width of the Lesions in Terms of the Energy Given Off 
Along the Bragg Curve 

Measurement of the width of the zone and the band of damage following 
exposin^e to 6,000 rad surface radiation dose was carried out in practically 
all 211 brains < Table I i. The average maximal width of the zone and of the 
band, both in the cerebellum and the cerebrum, was \irtually the same in 
the brains exposed to alpha particles as those exposed to protons (Table II) . 



386 



JANSSEN ET AL. 

TABLE I 



Number of Rat Brains Studied at Different Time Intervals Following 
Irradiation with Alpha Particles and Protons " 

Time of Dosage to brain surface (in rad) 

sacrifice 50 250 750 1,500 3,000 6,000 Totals 

after A AAA A A P 
irradiation 

Hours 1-6 — — — — — 8 2 10 

14-18 _____ 7_ 7 

20-28 _____ 13 4 17 

36-48 _____ 12 4 16 

Days 2.5-5 " _ _ _ 3 3 38 12 56 

6-11 _____ 35 25 60 

12-18 3 3 3 — — 17 5 31 

20-46 ____ 5 3 6 14 

64 43 3 3 3 9 — 25 

120 _____ 1 _ 1 

150 _____ 1 _ 1 

216 3 2 3 3 3 2 — 16 

10 8 9 9 14 153 58 261 

" Key: A, alpha paitirle radiation; P, pioton radiation. 



TABLE II 



Width of Region of Damage in the Cerebellu.m and Cerebrum Following 
Exposure to Alpha Particles and Protons at 6,000 Rad Surface Dose 



Site of irradiation 



Alpha particles 



Protons 



Zone (fji) Band (^) Zone (fj.) Band (^) 



Cerebellum 

average width 
Cerebrum 

average width 



6 hr-5 days 7-216 days 6 hr-6 days 7-30 days 
110 41 123 45 

24 hr-7 days 8-216 days 60 hr-6 days 7-30 days 
236 110 291 119 



On the other hand, the width of the zone and of the band was less for the 
cerebelkim than for the cerebrum by a factor of around 2/2- Due to the 
factor of tissue shrinkage as a consecjuence of irradiation, the width of the 
zone or band decreased with time. With respect to alpha particles, for ex- 
ample, the average width of the zone in the cerebellum during the 6 hour- 
5 day period was 110 /m. and at the 7-216 day period, 41 /x, at which time 
the "zone" had shrunk to form a "band."" In the brains exposed to a 3,000 
rad surface dose, the series was too small to obtain dependable averages. 
However, the width of the band was substantially less (Fig. 1 -4 ) . 



PATHOLOGIC CHANGES IN BRAIN CAUSED BY ALPHA PARTICLES 387 



:■•. ._.;:;*'^s^.gu^-^i?H%?f;;S,i'r'-'. • 




Fig. 1. Proton radiation. 6.000 rad surface dose; sacrifice at 10 days. A. A narrow 
cell-poor band extends across the cerebellar folia. Abo\'e the band the granular layer 
is rarefied, and some of the granule cells are atrophic. The intrafolial white matter is 
slightly disrupted and has reduced stainability. The Purkinje cells in the region of the 
band ha\e disappeared. Bergmann cells show little alteration. Proliferated glia are 
present in the molecular layer, especially at the le\el of the band. B. In the cerebral 
cortex a wider ner\e-cell-poor band is to be seen. Glia within the band ha\e increased 
somewhat in number. Pyknosis of the nerve cells near the cerebral surface represents 
postmortem artifact. Both stained by \'an Gieson-hcmatoxylin. Both X ~5. 



At the 6.000 rad surface i Fias. 1 and 2 ' a wide nene-cell-poor band was 
present in the cerebellum and cerebrum, but after 64 days both the band and 
the cerebellar and cerebral tissue abo\e the band were oreatly shrunken as 
compared with sections obtained at the 10-day stase. At the 3.000 rad surface 
dose, after 64 davs i Fia;. 3) the degree of damase was tar less ad\anced. At 














■.x<'-v 



^^-ll^^tSfe-?^'-''-- 



•>... 






^f^ 




J^''" 



Fig. 2. Alpha particle radiation, 6,000 rad surface dose; sacrifice at 64 days. A. A 
narrow band poor in granule cells lies just above an intact granular layer. Above the 
band is a wide zone in which practically all granule cells are shrunken. Not only is 
there cellular shrinkage, but there is also gross atrophy of the granular and molecular 
layers in the irradiated region. Many Purkinje cells have disappeared. Glia in the 
molecular layer have multiplied, and at the level of the band near the middle of the 
photograph the cells of the molecular layer have disappeared. The white matter in the 
irradiated area is pallid, and its glia ha\e increased somewhat in number. B. A nerve- 
cell-poor band, which contains an excess of glia, stretches across the cerebral cortex. 
The overlying cortex is greatly shrimken when comparison is made with that in Figs. 
1 and 3 of the same magnification. Below the hand are the white matter, ventricle, 
and hippocampus. C. From a field in the cerebral cortex, showing an atrophic band in 
which vessels are greatly dilated. The dark structures are vessels which have under- 
gone advanced hyalinosis. All stained by Van Gieson-hematoxylin. A and B, X 70; C, 
X 265. 

388 



PATHOLOGIC CHANGES IN BRAIN CAUSED BY ALPHA PARTICLES 389 



*■::*£ 5'i) 










i^:^'f 



fS 



.'-''^'■';^"^^^-'3% 









'.^igi^i^lii^^^^ 



Fig. 3. Alpha particle radiation. S.UOO rad surtace dose; sacrifice at 64 days. A. 
Cerebellum, showing a zone of shrunken granule cells marked oflT by a sharp lower 
border. Rarefaction of the granular layer is greatest in the region of ma.ximal depth of 
the damage. To the left, Purkinje cells in the region of most intense irradiation have 
disappeared. Glia in the molecular layer ha\e imdergone slight proliferation. B. Cere- 
brum. The nerve-cell-poor band of damage is marked off by a sharp lower border. 
.\bo\e the band, scattered nerve cells ha\e disappeared. Both X 65. Van Gieson- 
hematoxvlin stain. 



the 1,500 rad radiation level (surface dose), the band of damage, present 
only in the cerebrum, was significantly narrower at 216 days ( Fig. 4 i than at 
64 days after exposme to 3,000 rad. 

One cerebellum was studied with a view to determining whether the 
width of the zone of damage was consistent with the stopping power of the 
particles as expressed bv the Bragg cin\e. The ceiebellum had been exposed 
to a surface dose of 12,000 rad, and the animal was sacrificed at 24 hours. 
The lower border of the granule cell pyknosis (Fig. 5) was 720 deep to the 
cerebellar surface. The pyknotic granule cells were counted in 8 diflferent 
tissue depths within a 9 X 9.8 cm area on a photograph enlarged 225 times; 
the number of pyknotic cells for each of the 8 units within the area, when 



390 JANSSEN ET AL. 







'• . 






i 


1* 


'^ 

.« 


«» 


: ''' 


y\':^'^ ' 












% 


* 

^ 


'■ -^%'^' 




■ ■• , / . ,. 




'■• 




'\-^^ 




% 


:#:^ 








" " 






y 


f 


■■■^y-' 


vS'l ; 






■. 




-"• t 




% 






\ >: ; 


, f 


■,,i'' 






T^, 






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f ^ X 


« 


r 












B 






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Fig. 4. Alpha particle radiation, 1,5UU rad surface dose; sacrifice at 216 days. 

A. Cerebral cortex, illustrating a faint nerve-cell-poor band (arrows) extending later- 
ally from the region of interhemispheric fissure. The meninges appear unaffected. X65. 

B. Lamina in the region of the interhemispheric fissure. Numerous nerve cells have 
disappeared and others are faded. Two nuclei, presumably those of nerve cells, are 
enormously enlarged. X410. Van Gieson-hcmatoxylin stain. 

plotted against the Brago curve, coincided with the energy given ofT along 
the slopes and at the peatc of the curve (Fig. 6) if the assumption could be 
made that the cerebellum had undergone linear shrinkage by a factor of 
35% as the result of processing. Measurements were made to test this as- 
sumption. The maximal width of the cerebellum in the stained section in 
which the pyknotic cells were counted was 7,590 ju,; when this figure was in- 
creased by 35%, the value was 11,660 /x. In a control rat of the same age as 
the experimental animal, the maximal width of a fresh cerebellum immedi- 
ately after autopsy was 10,120 fx. Thus, the assumption was approximately 
correct. As to the cerebrum, its width on the stained section was 7,084 /a, 
and in the imfixed fresh brain, 8,602 ix. Thus, linear tissue shrinkage was 
relatively less for the cerebrum than the cerebellum. 

Alterations in Structural Elements with Respect to 
Time-Dose Relationships 
Nerve cells 

At surface doses of 50, 250, and 750 rad, observations up to 216 days 
(Table I) failed to re\eal any neiuonal changes. The lowest surface dose at 
which alterations were seen was 1,500 rad, and then not until the 216 day 
stage. In the cerebral cortex at this stage, a faint, narrow nerve-cell-poor 
band was detected (Fig. 4A) . Adjacent to the interhemispheric fissure, the 



PATHOLOGIC CHANGES IN BRAIN CAUSED BY ALPHA PARTICLES 391 






^^H^^^^ . 







Fig. 5. Alpha particle radiation, 1L!,000 rad surface dose: sacrifice at 24 hours. 
A fairly sharp border separates irradiated from nonirradiated granular layer. Above 
this border the density of pyknotic granule cells decreases progressively. The Purkinje 
cell (arrow) at the lower border of the damaged granular layer, i.e., in the most 
intensely irradiated region, shows an alteration in nuclear chromatin. Cells in the 
corresponding region of the molecular layer appear unaffected. Vessels are not dilated. 
X175. 



nuclei of occasional cells, piesiimablv nerve cells, were extraordinarilv en- 
larged ( Fis;. 4B ) . By contrast, no exidcnce of damage was obser\ed in the 
cerebellum. 

At the 3,000 rad sinface dose, with sacrifice at 4 to 216 days i Table I), 
two brains studied at 20 davs had a narrow band of "ranule cell rarefaction 



392 



JANSSEN ET AL. 




200 400 600 800 1000 

Tissue depth .microns 



1200 



Fig. 6. Distribution of pyknotic cerebellar granule cells (illustrated in Fig. 5) as a 
function of depth, superimposed on the physically measured Bragg curve, assuming 
linear tissue shrinkage of 35 9f. This assumption was fairly well substantiated by 
direct measurements of fresh cerebellum and of the stained section. 



in the cerebellum (and scattered pyknotic granule cells above the band) and 
a significantly wider band of ner\e cell damage or loss in the cerebral cortex. 
In the cerebellum in the region corresponding to the Bragg peak, the nuclei 
of occasional Purkinje cells were chromatin-poor, while others had under- 
gone the homogenizing type of necrosis; the molecular layer had a somewhat 
disrupted appearance. 

At the 6,000 rad surface dose a distinct zone of nerve cell damage was 
first encountered in the cerebellar cortex, namely at 6 hours in 4 of 6 
animals exposed to alpha particles or protons, and it consisted of a fairly 
even row of pyknotic granule cells in pale, somewhat loculated tissue (Fig. 
7A). No alterations were seen in Purkinje or Golgi cells or in nerve cells of 
the molecular layer. The earliest time interval at which nerve cell damage 
was encountered in the cerebral cortex was also 6 hours, but only isolated 
nerve cells were affected, chiefly in the region adjacent to the interhemi- 
spheric fissure. Such changes were foimd consistently in the first 2 days, and 
were virtually limited to the region corresponding to the Bragg peak. Some 
of the aflFected ner\e cells exhibited the homogenizing type of necrosis (Fig. 
7B) ; others, fading of nuclear chromatin, often advancing to complete nu- 
clear "skeletonization" ( Fig. 7C ) . Cellular damage at these early stages 
made it diflScult to determine whether some of the elements implicated were 
nerve cells or glia ( Fig. 8 ) . Both cell types appeared to be affected simulta- 
neously. The earliest time at which a distinct zone of damage was found in 
the cerebral cortex was 42 hours, but such damage, which was in the form 
of fairly broad interrupted segments, was found in only 1 of 4 brains studied. 
Some areas within the zone were spongy, and ner\'e cells showed the homog- 
enizing type of necrosis; in nonspongy areas within the zone of damage, the 



PATHOLOGIC CHANGES IN BRAIN CAUSED BY ALPHA PARTICLES ;393 



"VW^ 




Fig. 7. Ner\'e cell changes at early stages following irradiation at the 6,000 rad 
surface dose level. A. (6 hr) .\lpha particle radiation. Cerebellum, showing a fairly 
broad row of pyknotic granule cells in the region corresponding to the Bragg peak. 
Purkinje. Golgi. and Bergmann cells at this level appear unaltered. X675. B. (6 hr) 
Proton radiation. Cerebral cortex, showing a pyknotic cell, judged to be a ner\e cell 
which has undergone advanced necrosis. X675. C. (24 hr) Alpha particle radiation. 
Cerebral cortex in the region of the interhemispheric fissure (which is to the left, 
outside the field illustrated). Some ner\-e cells ha\'e \anished. and those that remain 
exhibit a varying reduction in the amount of nuclear chromatin. Some of the nuclei 
are "skeletonized." X375. \'an Gieson-hematoxvlin stain. 



. a>^''^>»«!^SK' 



W^ 




* \ 



-^(|P^ 



'i 



% 




4^ 



^ 



# 






B 



f 




•SMl' 



^ "'li 



Fig. 8. Damaged cellular elements in the cerebral cortex at relatively early stages 
following alpha particle radiation at 6,000 rad surface dose level. A. (6 hr) Nuclear 
breakdown in satellite glial cell. X720. B. (12 hr) Chromatin particles of disintegrated 
cell of undetermined nature. X 1170. C. (16 hr) Severely damaged nerve cells and 
glia in loosened tissue. Some of the pyknotic elements may have been as large as the 
nerve cell and its glial satellite in the upper right corner of the photograph. The large 
clear nucleus with the constriction is probably that of a damaged nerve cell. X900. 
D. (42 hr) Severely damaged cellular element. X1170. E. (8 days) Two mitotic 
figures are to be seen. X440. Van Gieson-hematoxylin stain. 

394 



PATHOLOGIC CHANGES IN BRAIN CAUSED BY ALPHA PARTICLES 395 



•4^ -^^^^^.^ 











I VI 




T U ^' Jl^ 



Fig. 9. Proton radiation. 6.(HJ0 rad surface dose; sacrifice at 30 days. Cajal impreg- 
nation of the cerebral corte.x. showing a wide ner\e-cell-free band through which apical 
dendrites are coursing. In the region of their passage through the band, some of the 
dendrites have decreased affinity for gold chloride and some appear somewhat swollen. 
Scattered reactive astrocytes are to be seen. X220. 



''skeletonization" type of ner\e cell necrosis pre\ailecl. Nerve cells abo\e the 
band had a strikinoly decreased affinity for tiold chloride, as did some den- 
dritic processes traversino- the band 1 Fia,. 9). 

From these data, a time-dose relationship in the development of lesions 
emerged (Table III). At the 1,500 rad surface close the feature that stood 
out was the presence of a nerve-cell-poor band in the cerebrum, but not 
in the cerebellum. At a smface dose of 3 /)00 rad both the cerebelhnn and cere- 
brum contained a distinct zone of nerve cell damage at 20 days. Precise time of 
initial damage at this dose level could not be ascertained because of lack of ma- 
terial between the 4 day and 20 day stages. At a surface dose of 6,000 rad, 
necrotic nerve cells were observed in the cerebellimi and cerebral cortex 
beginning at 6 hours after irradiation. In the cerebellum at the 6 hour stage 
a zone of granule cell pyknosis had been established, but it was not until 
the 42 hoiu- stage that a zone of nerve cell damage was encountered in the 
cerebrum. At the 12,000 rad surface dosage, nerve cell necrosis was evident 



396 



JANSSEN ET AL. 



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PATHOLOGIC CHANGES IN BRAIN CAUSED BY ALPHA PARTICLES 397 

in the cerebellum and cerebrum at 3 hours and a zone of damae:e at 4 hours. 
At these two times many more nerve cells were necrotic at 12.000 than at 
6,000 rad exposure. 

Glial lulncrability and reactivity 

Van Gieson-hematoxylin preparations were available from material ex- 
posed to all the dose levels: metallic impregnations, only from the 6,000 rad 
surface dose. For ob.servations on astrocytes, Cajal's gold chloride method 
was employed, and for the study of oligodendroglia and microglia, Penfield's 
modification of Hortega's silver carbonate technique was used. 

In Van Gieson-hematoxylin preparations from brains exposed to the 1 ,500 
rad surface dose of alpha particles, no glial changes were evident (Fig. 4). 
At 3,000 rad, enlarged astrocytic nuclei were noted in the damaged zone of 
the granular layer of the cerebellum at the 20 day period, and, at the same 
level, occasional Bergmann glia were faded and a few hyperplastic glia were 
evident in the somewhat disrupted molecular layer. In the zone of damage 
in the cerebrum, glia had obviously increased in number, and focal collec- 
tions of polymorphous reacti\e cells were seen here and there. At subse- 
quent stages glial reaction was more pronounced. 

At the 6,000 rad surface dose of alpha particles and protons, damage to 
glial cells was noticeable in the cerebellum in Van Gieson-hematoxylin prep- 
arations around the 4th day. consisting of chromatin loss in the nuclei of 
Bergmann cells and in cells of the molecular layer in the region correspond- 
ing to the Bragg peak. Subsequently, in the region of the upper slopes ot the 
Bragg cui-ve, glia in the molecular layer, and. in less measure, those ot the 
Bergmann layer, underwent multiplication Figs. lA and 2A). Glia in the 
region of the Bragg peak sometimes disappeared. Glia had trequently multi- 
plied in damaged intrafolial white matter as well. In the cerebral C(>rt( x in 
the region of the anticipated Bragg peak area, damage of isolated satellites 
and other glia was visible at various stages from 6 hours onward, and at later 
stages mitosis was evident i Fig. 8E i . 

In Cajal preparations from brains exposed to 6.000 rad surface dose the 
first changes in astrocytes in the cerebellum and the cerebrimi were noted 
at the 48 hour stage. Astrocytes in their entirety were enlarged and had an 
increased affinity for the gold chloride. In the cerebellum these changes 
were visible in the irradiated region of the cortex and in the intrafolial white 
matter, and later beneath the band, where Bergmann cells as well as astroglia 
exhibited prominent hypertrophy (Figs. lOA and lOB). In the cerebral cor- 
tex in early stages, astroglial reaction was particularly conspicuous in the 
region adjacent to the interhemispheric fissure. Subsequently, the reaction 
increased in intensity, and around the 3rd or 4th day a nerve-cell-poor band 



398 



JANSSEN ET AL. 






V-*^* X''^'^ 



A 



v'* 






^<A^_ 










Fig. 10. Alpha particle radiation, 6,000 rad surface dose. Cajal preparations of 
cerebellum. A. (9 days) Above the line separating irradiated from nonirradiated cortex, 
the cerebellar tissue has lost its affinity for the gold chloride. Beneath this line, many 
enlarged astrocytes are to be seen in the granular layer, and Bergmann cells have 
undergone hypertrophy. X80. B. (10 days) The hypertrophied Bergmann cells are to 
be seen to better advantage. X 150. 



became apparent ; within the band the astrocyte.s were undergoing distinte- 
gration. At 5 days, astrocytes had disappeared from the band and some 
above the band were in a state of advanced distintegration (Fig. IIA). Im- 
mediately beneath the band, the astrocytes were strikingly hypertrophic and 



PATHOLOGIC CHANGES IN BRAIN CAUSED BY ALPHA PARTICLES 399 






,s 






■ 3 . rSrS 













"i^'--*S? 










-%v: 






Fig. 1 1. Legend on next page. 






400 JANSSEN ET AL. 

had long- processes which extended up to the lower edge of the band. Sub- 
sequently, the astroglial reaction was present in the subcortical white matter 
and corpus callosum, particularly in the region adjacent to the interhemi- 
spheric fissure (Fig. IIC). In later stages after irradiation (up to 5 months), 
the zone of tissue destruction became increasingly narrowed and was de- 
void of astrocytic elements; however, along the upper and lower surfaces of 
the zone hypertrophic astrocytes persisted. 

Oligodendroglia in both cerebellum and cerebrum were diflficult to im- 
pregnate. Data on their vulnerability are, thus, not available. 

Reactive changes of microglia in the cerebellum and cerebrum in Hortega- 
Penfield preparations were first noted at 48 hours after irradiation. In the 
cerebellum at this time there were scattered reactive microgliacytes in the 
cortex. Subsecjuently, they were noted over a wide irradiated area (Fig. 12A) . 
A similar microglial response was observed in the cerebral cortex, being 
particularly evident in the white matter beneath the mesial cortex, i.e., in 
the region adjacent to the interhemispheric fissure. At earlier stages hyper- 
trophied microglia were noted here and there at various levels of the cortex. 
With the development of a band of nerve cell damage, microglia appeared 
within the band as well as in the underlying cortex and white matter. 
Changes in the cerebral cortex at the 4 day stage are illustrated in Fig. 12B. 
Subsecjuently, most of the microglia were in a hypertrophic state and occa- 
sionally appeared ameboid. Gitter cell forms were few. After the first week 
following irradiation, the zone usually contained microglial cells which, 
when their cell body was round, could be distinguished from oligodendro- 
cytes by the short thorny spikes on their main cytoplasmic processes (Fig. 
12C). In adjacent areas, most conspicuously just above the band, diffusely 
scattered hypertrophic cells, including rod cells, were noted. 

Mesenchymal elements 

In general, both histiocytic and fibroblastic reactions occurred in the 



Fig. 11. Alpha particle radiation, 6,000 rad surface dose. Cajal preparations of 
cerebrum. ^. (5 days) In the band (the clear region in the middle of the photograph), 
there are occasional necrotic nerve cells and astroglia. In the part of the zone about 
the band, nerve cells have vanished or are in a necrobiotic state ; astrocytes, which are 
hypertrophic, are disintegrating. Beneath the band the astrocytes are hypertrophic 
and have prominent processes oriented toward the band. X335. B. Astrocytes in the 
corresponding area of a normal control rat. X400. C. (16 days) A fairly wide band 
extends across the cortex. In the middle of the photograph some nerve cells beneath 
the band are damaged, and hypertrophied astroglia are to be noted from the level 
just beneath the band down to, and including, the corpus callosum, where an area of 
the white matter is pallid. Vascular dilatation is most prominent in the region of the 
zone and the band just above the area of damage beneath the band. X44. 



PATHOLOGIC CHANGES IN BRAIN CAUSED BY ALPHA PARTICLES 401 




Fig. 12. Legend on next page. 



402 JANSSEN ET AL. 

meninges and in the sheaths of larger vessels in the meninges and in the 
cerebellar and cortical substance; the histiocytic phase occurred earlier and 
the higher the dose, the more pronounced it was; the reactions were usually 
most advanced in the region corresponding to the Bragg peak, and in time 
they advanced upward in accordance with the irradiation dose. After a con- 
siderable latent period following exposure to the 3.000 and 6.000 rad sur- 
face doses, blood vessel walls frequently underwent fibrohyaline change or 
hyalinosis (Fig. 2C). 

Circulatory and Vascular Changes as Brought Out by Stains 

Sections from 83 brains exposed to alpha particles and from 30 exposed 
to protons, all at the 6,000 rad surface dose level, were stained by the Pick- 
worth-Lepehne method from 1 hour to 5 months after irradiation. 

In sections prepared by routine methods, no vascular changes, hemor- 
rhages, or plasma transudates were encountered in brains exposed to surface 
doses of 50, 250, 750, 1,500, or 3,000 rad. At 6,000 rad surface dose, the 
v^arious circulatory and vascular changes produced by alpha particle and 
proton irradiation were the same, whether in the vessels of the cerebellum 
or cerebrum. At various times through 18 hours, no vascular dilatation was 
observed within the region presumably irradiated. It was noted in a rather 
wide zone of irradiation in both cerebellum and cerebrum at 24, 36, and 
42 hours. 

In Pickworth-Lepehne preparations, the earliest dilatation of vessels ob- 
.served in the cerebellum and cerebrum was at 48 hours. It was diffuse in 
the irradiated region from the pia downward. By 2.5 days, it had become 
concentrated in the region corresponding to the upper slopes and the peak 
of the Bragg curve. The lower border of the zone of vascular dilatation was 
fairly sharp and corresponded to the lower border of the zone of tissue 
damage, as seen in Van Gieson-hematoxylin preparations. At 3 days, the 
cerebellum and cerebrum in 1 of 3 rats contained tiny hemorrhages predom- 
inantly in the "'Bragg peak" area. At 4 days, capillaries within the band had a 
suggestively swollen basal membrane, while others had collapsed. Within the 



Fig. 12. Radiation, 6,000 rad surface dose. Hortega-Penfield preparations. A. (30 
days) Proton radiation. Cerebellum. The maximal depth of irradiation is at the level 
of the highest Purkinje cell. Microgliosis has occurred in the granular layer, intrafolial 
white matter, and the molecular layer, and Bergmann cells are hypertrophic. XI 10. 
B. (4 days) Alpha particle radiation. Cerebral cortex. The light area at the top of 
the photograph is the band of damage. Beneath it are increased numbers of hyper- 
trophic microgliocytes. X310. C. (30 days) Proton radiation. Region of interhemi- 
spheric fissure of cerebral cortex, showing a fairly wide band of damage in which 
great numbers of microgliocytes are congregated. X 145. 



PATHOLOGIC CHANGES IN BRAIN CAUSED BY ALPHA PARTICLES 403 

band of damage there were minute hemorrhages and extravasates of globu- 
lar proteinaceous material. Between 5 and 8 days, vascular dilatation was 
more evident (Fig. 13A), and a fair number of reactive cells had appeared 
in the sheaths of vessels in the nerve-cell-poor band and somewhat higher. 



V 



>-.\- 



,>- 



*S^ 



# 



IN 



-*. ^ 




Fig. 13. Radiation. 6.000 rad surface dose. Pickworth-Lepehne preparations. A. (8 
days) .\lpha particle radiation. \'ascular dilatation is to be seen in the irradiated part 
of the cerebellum and cerebrum, and in the latter is most apparent in the region cor- 
responding to the peak and upper slopes of the Bragg cur\e. X6. B. (9 days) Proton 
radiation, .\neurysmal vascular dilatation and multifocal lack of erythrocyte staining 
are to be noted. Occasional hemorrhages ha\e occurred. X50. 



404 JANSSEN ET AL. 

Between 9 and 16 days, there were more filling gaps in the vascular tree in 
the irradiated zone (Fig. 13B), more evident degenerative changes in the 
walls of smaller vessels, fibrinoid necrosis and swelling in the walls of occa- 
sional arterioles in the band, and fusiform and saccular microaneurysms. 
During these stages the hemorrhages increased in number and size and had 
a linear arrangement in the band of cerebellar and cerebral damage. Hemor- 
rhages were occasionally seen below the band. After 18 days, they were 
rare. In the brains of 2 animals which survived 30 days, the capillary net- 
work in the band was as rich as in the adjacent cortical tissue. Vessels just 
above and just below the band were still dilated. At 4 and 5 months, at a 
time when the band was greatly shrunken, the same vascular conditions 
prevailed. 

Vascular Permeability to Fluorescein-Labeled Serum Proteins 
(FLSP) AND Sodium Fluorescein 

Altogether 54 rats (38 for FLSP and 16 for sodium fluorescein studies) 
were exposed to a 6,000 rad surface dose of alpha particles. FLSP is innoc- 
uous and imder normal conditions does not penetrate vessel walls. In the 
cerebellum, penetration of vessels by FLSP was first noted at 72 hours in 1 
of 3 animals studied. The disturbance of vascular permeability was striking 
in that intrafolial white matter below the Bragg peak zone showed extensive 
green fluorescent mottling. In the granular layer within the irradiated zone 
and below the zone, widespread glial cells contained FLSP inclusions, and 
in the molecular layer at corresponding levels small FLSP globules were 
foimd about a few capillaries. The final stage at which increased permeation 
of FLSP was visible in the cerebellum was in an animal sacrificed 18 days 
after irradiation. 

In the cerebrum the first microscopically detectable vascular penetration 
by FLSP was at 48 hours and consisted of small, bright-green fluorescent 
droplets of FLSP along the outer surface of a few capillaries in widely 
scattered areas of the exposed cerebral cortex (Fig. 14). At 72 hours the 
capillaries exhibiting increased permeability to FLSP were much more nu- 
merous in the cortex, and many neuroglial cells in the imderlying white matter 
were studded with FLSP inclusions. One cerebrum showed an irregular 
green fluorescence throughout the imderlying white matter. Subsequently, 
intraparenchymal FLSP was conspicuous aroimd the blood vessels in the 
cortex, especially those in the zone. In the underlying white matter, protein 
inclusions were found in the glial cells, and in some regions the white matter 
exhibited irregular, mottled FLSP fluorescence. FLSP transport occurred to- 
ward the pial and ependymal smfaces, as judged by the collection in these 
regions of protein inclusions. In the cerebrum of 1 of 2 rats sacrified at 36 



PATHOLOGIC CHANGES IN BRAIN CAUSED BY ALPHA PARTICLES 405 




Fig. 14. Alpha particle radiation. 6,000 rad surface dose: sacrifice at 48 hours. The 
animal was injected with 2 cc of 8''f fluorescent albumin 24 hours before sacrifice. 
A capillary in the irradiated corte.x, showing droplets of extravasated fluorescent al- 
bimiin. .Approximately x400. 



days, the outer surface of a few small blood vessels in the cortex was still 
studded with FLSP droplets. The superficial leptomeninses contained nu- 
merous FLSP-laden macrophages. In animals sacrificed at 4 and 5 months, 
no abnormal permeation of FLSP was noted. 

The first appearance of FLSP fluorescence in the f^ross brain was ob- 
ser\ed at 72 hours. At this stage the irradiated region of both cerebellum 
and cerebrum fluoresced. In coronally cut blocks of the cerebellum, the 
fluorescence was confined to the irradiated region of the folia. In the cere- 
brum a faint green fluorescence was noted in the irradiated part of the cor- 
tex and a brighter green fluorescence in the underlying white matter, in- 
cluding the corpus callosum. In the next jeie days the white matter below 
the irradiated medial cerebral cortex exhibited intense green fluorescence, 
particularly in the region close to the interhemispheric fissme. The cortex 
showed much less intense green fluorescence. In both cerebellum and cere- 
brum grossly visible fluorescence persisted for approximately a week and 
then diminished. Faint fluorescence was last seen at 13 days after irradiation. 



406 JANSSEN ET AL. 

The study with sodium fluorescein was limited to gross observations be- 
cause it does not locahze microscopically. Parenchymal fluorescence was 
first seen at 72 hours, and resembled that obser\ed with FLSP, except that 
the contrast in intensity in grey and white matter was much less distinct. 
The final stage at which fluorescence in both cerebellum and cerebrum was 
seen was 14 days after irradiation. 

Discussion 

The foregoing observations supplement in various ways those reported by 
Malis and his associates (1957 ) dealing with the efTects of protons ( 10 Mev 
per nucleon) on the cerebral cortex of 2 cats. Although, in terms of energy 
transfer, comparison of results meets obstacles because of the difficulties 
they encountered in establishing tissue irradiation dosage, the pseudolaminar 
cerebral cortical lesion they produced was highly similar to that observed 
in our animals. 

One of the problems with which we were concerned was whether or not, 
at a given radiation dosage, alpha particles produced the same pathologic 
changes as protons in the cerebellum and cerebrum. Although alpha par- 
ticles and protons with the same energy per nucleon (in the present investi- 
gation, about 12 Mev per nucleon) have the same range, the stopping 
power, or linear energy transfer (LET), of the alpha particle is 4 times 
that of the proton. It was therefore of interest to determine whether the 
fourfold difference in LET was reflected pathologically. At the 6,000 rad 
surface dose, the changes produced by these two types of particles were 
highly similar in the time of appearance of lesions, width of the band of 
most intense damage (Table II), extent and severity of nerve cell damage in 
the tissue above the band after a given latent period, and time of appearance 
of vasodilatation in sections stained by the Pickworth-Lepehne method 
(Table III). Thus, pathogenically, alpha particles and protons had much 
the same effect despite the fourfold difference in particle LET. 

In animals exposed to the lowest effective radiation dosage (1,500 rad at 
the brain surface), a band lesion was evident in the cerebrum at 216 days 
(Fig. 4), when no changes were observed in the cerebellum (Table III). 
This was considered as evidence that the cerebral cortex was the more 
radiovulnerable. At 6,000 rad cerebellar granule cells and nerve cells and 
glia of the cerebral cortex seemed equally radiovulnerable from the stand- 
point of the time of appearance of damage, but a zone of damage was evi- 
dent earlier in the cerebellum (6 hours) than in the cerebrum (42 hours). 
Invariably the band of nerve cell loss in the cerebellar granular layer was 
much narrower than that in the cerebral cortex (Table II). Whether this 
means that nerve cells of the cerebral cortex were more radiovulnerable than 



PATHOLOGIC CHANGES IN BRAIN CAUSED BY ALPHA PARTICLES 407 

the oranule cells of the cerebellum is not clear to iis. A further point is that 
with the passage of time the tissue abo\"e the zone of damage in both cerebel- 
lum and cerebrum underwent profoimd atrophy i Figs. 1 and 2 ) . Measure- 
ments indicated that reduction in the width of the irradiated part of the 
cerebellum was relatively somewhat greater than that in the cerebrum 
'Table II i. This does not necessarily mean that the cerebellum was rela- 
tively more radio\ulnerable. for the cerebellum underwent greater shrinkage 
than the cerebrum during processing of the brain. 

Nei*\"e cells and neuroglia of the cerebral cortex appeared equally vulner- 
able at early stages, as indicated at the 6,000 rad surface dose levels by 
necrosis of isolated nerve cells and glia (Figs. 7 and 8), and at subsequent 
stages, as indicated by necrosis of astrocytes and nerve cells (Fig. IIA). In 
the cerebellum the granule cell seemed the most radiovulnerable. Depending 
on dosage and time, \arying numbers of these cells in the more intensely 
irradiated part of the granular layer subsequently underwent necrosis. 
Purkinje cells situated at comparable levels in the cerebellum also suffered, 
but the tempo at which the damage occurred seemed slower than that in 
granule cells. Purkinje cells .sometimes appeared intact at a time when 
granide cells were pyknotic (Fig. 7A). Since at later stages following irradi- 
ation the width of the zone of Purkinje cell damage was often not much dif- 
ferent from that of the pyknotic granule cells (Figs. lA, 2A, 3 A, and 12A), 
it was concluded that relatively little difference in radiovulnerability of these 
two cell types existed. Less radio\ulnerable than granule and Purkinje cells 
were Bergmann cells, nerve cells and glia of the molecular layer, and Golgi 
cells in the granular layer, in that order of decreasing radiovulnerability. At 
fairly early stages, damage was also incurred by intrafolial white matter, and 
the reacti\ ity of its glial cells was about the same as that elsewhere. 

E.xamination of material exposed to the 1,500 rad surface indicated that 
nerve cells had been destroyed and that blood \essels were not morpho- 
logically altered (Fig. 4). The same was true at the 3,000 rad level at 20 
days when a zone of cytologic damage was encountered. At 6,000 rad, nerve 
cell and glial necrosis occurred as early as 6 hours after irradiation, but it 
was not until 48 hours that vasodilatation as brought out by the Pickworth- 
Lepehne method appeared and not until 60 hours that vasodilatation was 
concentrated in the '"Bragg zone."" Clearly, ner\e cell and glial damage oc- 
curred before circulatoiy distinbances sufficient to cause vasodilatation were 
e\iclent. Moreover, only at 48 hours did the blood-brain barrier become per- 
meable to FLSP, indicating that during the preceding hours any vascular 
change that might ha\e occurred was not functionally evident. Further, the 
presence of hemorrhages, taken as e\idence of vascular damage, did not 
occur until the 3rd day alter irradiation. The conclusion seems inescapable 
that, in earlier stages at least, nerve cells and glia were primarily damaged 
by particle radiation. Damage of nerve cells and glia concurrently is most 



408 JANSSEN ET AL. 

clearly shown in Fi^. 8C. From their study of the brains of mice exposed to 
a 25-/i,-wide beam of deuterons with an energy of 22.5 Mev per nucleon, 
in which the width of the track corresponded to the width of our bands, 
Zeman et al. (1959) reached the conclusion that damage to nerve cells and 
glia represented a direct irradiation efTect. 

Further evidence pointing to primary cellular damage in our animals, 
which would not be expected if circulatory disturbance and an edematous 
process were the sole factors, was the close correlation of the granule cell 
pyknosis with the magnitude of the energy given off" along the slopes and at 
the peak of the Bragg curve (Figs. 5 and 6) and the straightness of the lower 
border of the irradiated zone. In later stages following exposure to particle 
radiation (i.e., from 5 or 6 days onward), it would be difficult to judge 
to what extent circulatory disturbances contributed to the extent of the 
damage. There were two pieces of evidence that circulatory disturbances 
did contribute to further advance of lesions. In sections stained by the 
Pickworth-Lepehne method (6,000 rad surface dose), collapse of occa- 
sional small vessels in the band of cellular damage was noted at 4 days, 
and numerous filling defects were found in the vascular tree from the 9th 
day to about the 24th day. Secondly, lesions in the cerebral cortex were 
particvdarly striking in the region of the interhemispheric fissure (Figs. 4, 
7C, and IIC), and cortical nerve cells below the band in this general region 
were sometimes damaged in association with astrogliosis and microgliosis, 
which extended down into the subcortical white matter and corpus callosum. 
Such effects were considered to be related to greater circulatory disturbances 
in this region than elsewhere, perhaps because in this "angle" the vascular 
tree is more prone to be constricted by a generalized edematous process 
than elsewhere. 

With regard to the blood-brain-barrier permeability studies, the FLSP 
technique had the advantage over the sodium fluorescein indicator in that the 
labeled proteins could be demonstrated microscopically both before and 
after the gross fluorescence was perceptible (Table III). Extension of the 
fluorescence to regions far beyond the irradiated area was considered to 
have been due to disturbance of vascular permeability initiated in the irra- 
diated area. Even ependymal epithelial cells lining the upper wall of the 
ventricle contained large quantities of fluorescent protein. 



Summary and Conclusions 

This article deals with the effects on the cerebellum and cerebrum of 
alpha particles with an energy of about 12 Mev per nucleon at surface doses 
of 50 to 6,000 rad, and of protons of the same energy at a surface dose of 
6,000 rad. The dorsal surface of much of the cerebellum and cerebrum was 



PATHOLOGIC CHANGES IN BRAIN CAUSED BY ALPHA PARTICLES 409 

exposed. The maximal tissue dosage was approximately 5 times that at the 
brain surface. 

No essential differences were observed in the alterations produced by- 
alpha particles and those produced by protons. 

The width of the zone of damage in the cortex of both the cerebelkan and 
cerebrum corresponded to the amount of energy gi\en off by the particles as 
expressed by the Bragg cinve. 

Granule and Purkinje cells of the cerebellum and nerve cells and glia in 
the cerebral cortex were highly radio\ulnerable. The greater width of 
the band of damage in the cerebrum than in the cerebellum and the observa- 
tion that at the lowest effective radiation dose (1,500 rad at the brain 
surface) a band of nerve cell loss was found in the cerebrum but not in the 
cerebellum suggested that the cerebral cortex was the more radiovulnerable. 
From other standpoints. howe\er, the cerebellar granular layer seemed the 
most radiovulnerable. 

Nerve cell and glial damage was incurred before circulatory or perme- 
ability disturbances were evident, strongly suggesting direct irradiation of 
cellular elements as the primary factor in pathogenesis. At subsccjuent stages 
circulatory changes and increased vascular permeability contributed to the 
extent of the lesions. 

No lesions were encountered after as long as 7 months at surface doses of 
50, 250, and 750 rad. The earliest times at which damage to cellular elements 
was obserxed following exposure at other siuface doses were as follows: at 
1 ,500 rad. 7 months ( in cerebrum alone ) ; at 3,000 rad. 20 days; at 6,000 rad, 
6 hours; and at 12,000 rad, 3 hours. 

References 

Birge, A. C. .^nger. H. O., and Tobias, C. .■\. 1956. Heavy chargcd-particle beams. In 

"Radiation Dosimetry" (C. J. Hine and G. L. Browncll, eds.), pp. 623-665. 

Academic Press. New ^'ork, 1956. 
Brustad, T., Ariotti, P., and Lyman, J. 1960. Experimental Set-Up and Dosimetry 

for Investigating Biological Effects of Densely Ionizing Radiation, LTniversity of 

California Research Lab. Report, in press. 
Klatzo, I., Piraux, A., and Laskowski. E. J. 1958. The relationship between edema. 

blood-brain-barrier and tissue elements in a local brain injury. /. Neuropathol. 

Exptl. Neurol. 17, 548-564. 
Klatzo, I., and Miquel, J. 1960. Obser\ations on pinocytosis in nervous tissues. /. 

Neuropathol. Exptl. Neurol. 19, 475-487. 
Malis. L. I.. Loevinger. R., Kruger, L.. and Rose. J. E. 1957. Production of laminar 

lesions in the cerebral cortex by heavy ionizing particles. Science 126, 302-303. 
Simpson, M. E., Van Dyke. D. C, Asling, C. W.. and Evans, H. M. 1953. Regenera- 
tion of the calvarium in young normal and growth hormone-treated hypophysec- 

tomized rats. Anat. Record 115, 615-625. 
Zeman, W., Curtis, H. J., Gcbhard, E. L., and Haymaker, W. 1959. Tolerance of 

mouse brain tissue to high-energy deuterons. Science 130, 1760-1761. 



Some Observations on Radiation Effects on 

the Blood-Brain Barrier and Cerebral 

Blood Vessels* 

Carmine D. Clemexte 

University of California at Los Angeles Medical Center, and 

the V. A. Hospitals, Los Angeles and Sepulveda, California 

AND 

Harold E. Richardson, Tr. 

Donner Laboratory. Uni; ersity of California, Berkeley, California'^ 

For many years it has been supposed that the adult nerxous system is rela- 
tively resistant to radiation dosaces capable of injuring or destroyino cells 
of other tissues and ora;ans. The primary reason usually forwarded to ex- 
plain this phenomenon has been that the primitive cells in the body are 
more radiosensitive than cells differentiated to a hisih order of specializa- 
tion. In this regard obser\ations seem to su^cest the thesis that cellular 
radiosensiti\ity depends somewhat on the rate of nucleic acid synthesis. It is 
usually stated that lymphocytes, developing germ cells, and maturing neuro- 
blasts are among the most radiosensitive of cells, whereas the adult neuron 
is among those most resistant. 

When assessing the effects of radiations on the brain, it seems difficult to 
speak in generalizations as to the radiosensiti\"ity or radioresistance of the 
entire organ. For in the central ner\ous system, as in other organs, the 
pathology which develops with ionizing radiations can be considered to 
result from several sources or a combination of them. We describe paren- 
chymal, stromal, and intercelkdar effects of radiation in most other organs 
in the body. Therefore, it seems wise not to ha\e our opinions of the radio- 
sensitivity of the brain as an organ unduly influenced, just because the adult 
neuron has been described as being relati\ely radioresistant. As a matter 
of fact, even this latter generality has been questioned recently (Grigoriev 
and Tsypin, 1957; Lebedinsky ct al., 1959; Livanov and Biryukov, 1959: 

* These in\estigations were supported in pait by the U.S. Pubhc Health Service and 
in part by work under contract with the U.S. Atomic Energy Commission. 

t The authors wish to thank Dr. C. Tobias for his ad\ice and enthusiasm in the 
experiments dealing with high energy radiations. Mr. A. Huish for his photographic 
assistance, and Miss B. Kulgren and Mrs. J. Luce for their technical assistance. 

411 



412 CLEMENTE AND RICHARDSON 

Gangloff and Haley, 1960), and we have little information of subtle changes 
that radiation can produce in neuronal function or of neurochemical obser- 
vations. 

We would like to slant this report away from the direct effects of radia- 
tion on the neuron and examine more closely our research results, and those 
of other investigators, on the cerebral capillary and neuroglial systems. This 
we feel has an important place in such a conference because it has been 
shown that brain radiations can result in edema and inflammatory reactions 
around cerebral capillaries and because neurons are elaborately sensitive to 
ionic and other chemical changes in their environment; their functional in- 
tegrity can be altered by the slightest alteration in its metabolic medium. 

The Concept of the Blood-Brain Barrier 

That a selective barrier exists between certain circulating elements in the 
blood stream and brain tissue has been known since Ehrlich (1885) found 
that the brain remained unstained following the intravenous injection of 
acidic dyes, which brilliantly stained most of the other organs. This peculiar- 
ity in regard to the central nervous system has given rise to the concept of 
the existence of a blood-brain barrier, and in general it is observed that 
transvascular permeability characteristics tend to be manifested by a slower 
exchange rate of substances passing between blood and brain (in com- 
parison to other organs) rather than a faster rate of exchange. 

The acidic aniline dye, trypan blue, has been classically used by many in- 
vestigators to demonstrate the blood-brain barrier phenomenon. More re- 
cently, certain radioactive tracers (notably P'-, Br--, and Na^*) have been 
used. Although the use of dye techniques has been valuable in the formula- 
tion of the barrier concept, these methods are not above criticism. It should 
be pointed out that acidic dyes (such as trypan blue) tend to bind more 
completely with plasma proteins than basic dyes, which tend to stain brain 
structines following systemic administration ( Bennhold, 1932). Were we to 
depend on dye studies alone, it could be argued that the blood-brain barrier 
is merely a display of the impermeability of the cerebral vasculature to 
plasma proteins, a reasonable and predicable situation. 

With the advent of radioactive tracer methods, however, it has been shown 
that following the systemic injection of certain ions the ti'acer becomes dis- 
tributed throughout all the organs of the body uniformly and rapidly, 
whereas the relative concentration of the tracers in the brain are significantly 
lower (Manery and Bale, 1941; Greenberg et al., 1943; Wang, 1948). 
Although the rate of passage of radioactive tracers from blood to brain 
differs with respect to the particular ion being studied, curves can be ob- 
tained which reveal the amoimt of tracer in the brain at any given time 



EFFECTS ON BLOOD-BRAIN BARRIER, CEREBRAL BLOOD VESSELS 413 

after the systemic administration of the radioactive ion, and one can com- 
pare the amount of radioactivity in brain tissue with respect to other 
oroans in the body such as liver, spleen, or kidney. Thus, it is generally 
accepted today that the phenomenon of a blood-brain barrier exists. 

How the blood-brain barrier functions and the anatomic locus of the 
blood-brain barrier are more hypothetical. These two cjuestions are, by their 
nature, related. At present two groups of structures are considered by various 
investigators to be the possible anatomic sites of the blood-brain barrier: 
the capillary endothelium of the central nervous system and the perivascular 
sheath comprised of the membranes of the neuroglial cells. 

The proponents of the capillary wall theoiy point to many experiments 
performed with vital dyes. At first it was felt that the endothelial cells of 
cerebral vessels were different in their permeability characteristics because 
they remained unstained with vital dyes (Spatz, 1933). Broman (1937, 
1940a,b) also emphasized the vascular walls as being the site of the blood- 
brain barrier and noted that \itally stained vessels in the choroid plexus 
were in communication with branches within the brain which did not stain. 

Hauptmann and Gartner i 1932 ) . Hoff ( 1933 ) , and Tschirgi ( 1952) have 
expressed a preference for the perivascular glial membrane (Figs. lA and 
IB) as the more probable locus of the selective vascidar-permeability char- 
acteristics encountered in the brain. Although the capillary wall enthusiasts 
today still far outnumber the neuroglial membran-e supporters. Tschirgi 
(1958) offers a most interesting theory involving an active mechanism of 
transport of substances from blood to brain and metabolites from brain to 
blood, rather than a passive mechanism of diffusion. For an excellent review, 
sec the chapter written by Tschirgi > 1960) in the "Handbook of Physiology." 

Radiation and Blood-Brain Barrier 

It is well known that certain permeability changes in the cerebral \ascula- 
ture can occur following direct trauma to the brain, during simple exposure 
of the nervous system ( Macklin and Macklin. 1920; Prados et al., 1945; 
Grenell and McCawley, 1947), or following intravenous injection or topical 
application of certain chemical substances such as Diodrast ( Broman and 
Olssen. 1948, 1949; Bassett ct al., 1953), epinephrine ( Friedemann and El- 
keles, 1932). and hydrogen peroxide (Gi\re and Rexed. 1948). Thus, it is 
not surprisino- that another mode of physical injury to the brain, exposure 
to ionizing radiation, is also capable of producing permeability changes in 
cerebral \essels. This fact has been recognized since Rachmanow (1926) 
and Mogilnitzsky and Podljaschuk (1930) showed that ionizing radiations 
caused the perivascular neuroslia of mice and rabbits to be stainable with 
trypan blue. 



414 



CLEMENTE AND RICHARDSON 




Fig. 1. (A.) A section of monkey parietal cortex impregnated to show astrocytes by 
the Cajal gold sublimate method. Note the myriad numbers of astrocytic processes 
surrounding the capillary forming the so-called perivascular glial membrane. X 150. 
(B.) An electron micrographic demonstration of the perivascular astrocytic processes. 
Notice the virtually complete encasement of the capillary by the watery-looking cyto- 
plasmic projections of the nearby astrocyte. Approximately X 1,500. (Figure 1-B was 
loaned to the authors by Drs. D. Pease, E. Maynard, and R. Schultz. ) 



As is the case with the blood-brain barrier, pathologic, chemical, and 
physical insults are capable of breaking down the blood-aqueous humor 
barrier and the blood-cerebrospinal fluid barrier. The most obvious mani- 
festation of a breakdown in the blood-fluid barriers is an increase in protein 
in the central humor (Davson, 1960). Again, it has been shown that ex- 
posure to x-rays afTects the permeability characteristics of these blood-humor 
barriers. An increase in permeability of the blood-cerebrospinal fluid barrier 
was observed by Tatsumi as early as 1933 and by Hsu ct al. in 1936. 

In a series of 37 monkeys, Clemente and Hoist (1953, 1954) observed 
changes in permeability of cerebral vessels following single doses of x-irradi- 
ation ranging from 1,500 to 6,000 r. Trypan blue injected intraperitoneally 
demonstrated a profound functional impairment of the blood-brain barrier, 
especially in the brain stem and hypothalamus, and was best visualized after 
doses of 4,500 and 6,000 r (Fig. 2). Neuroglial, neuronal, and vascular dam- 



EFFECTS ON BLOOD-BRAIN BARRIER, CEREBRAL BLOOD VESSELS 415 




Fig. 2. A midsaggital \iew of the brain of a monkey which had recci\cd 4,500 r of 
x-irradiation (250 kw 118 r per min) to the head and which sur\'ived for 21 hours. 
Notice the trypan bhie discoloration in the medulla, hypothalamus, thalamus, and 
septum brought about by the radiation effects on the permeability of the blood-brain 
barrier. (Taken from Clemente and Hoist. 1954.) 



age (Figs. 3A-E ! was obser\able microscopically, and the areas of trypan 
blue penetration into the brain were characteristically those regions in 
which astrocytic damage was most intense. 

Monkeys subjected to a 1 .500 r head x-radiation dose and sacrificed 4 to 
8 months after irradiation showed pinpoint areas of degeneration surround- 
ing blood vessels in cortical and subcortical white matter (Fig. 4). The 
lesions were characterized by a.xon swelling, myelin breakdown, phagocytosis, 
and astrocytic scar gliosis. With higher doses of x-ray radiation to the brain 
in adult rats (20,000 to 23.000 v), Brishtman (1959) likewise found vascu- 
lar changes characterized by petechial hemorrhagic foci, and "the hem- 
orrhagic areas were stained a dark blue in those rats gi\en trypan blue."" 

Although obvious early changes in the permeability of the blood-brain 
barrier were difficult to obser\e with trypan blue in monkeys gi\en 1.500 r 
( Clemente and Hoist, 1953, 1954), it was felt that a more sensiti\e method 
of evaluating increases in the permeability of the blood-brain barrier might 
reveal such alterations. The study by R. G. Rose (1958) seems to have sub- 
stantiated these suspicions. He utilized the rate of passage of intravenously 
injected radioactixe sodium into the brain and cerebrospinal fluid of radi- 
ated and nonradiated rabbits to determine the influence of lower x-ray doses 
(^100 to 1.500 ri on cerebral capillary permeability. His data indicate an 



416 



CLEMENTE AND RICHARDSON 



{* % t / ' • . f * % ' 










^ • '^ - .• v';-- -•,•/-, 'Ki^-' 'JS-JiS^r:^. 






B 







1 






Fig. !3. In these 5 pictures are examples of some acute vascular and perivascular 
reactions in various areas of the brain after head x-irradiation in monkeys. (250 kv, 
half-value-layer for filtration of 0.5 mm Cu and 1.0 mm Al was 1.4 mm Cu; mid- 
cranial focal distance 35 cm; Dosage rate 118 r per min.) Thionin, H. and E., 
and Van Gieson stains. (A.) Petechial hemorrhage in white matter of occipital cortex, 
8 hours after 4,500 r. X 175. (B.) Perivascular leucocytic reaction in medulla 36 hr 
after 6,000 r. X 175. (C.) Perivenous reaction in the lateral hypothalamus 8 hr fol- 
lowing 4,500 r. X 140. (D.) Red and white blood cell diapedesis in the caudate nu- 
cleus in a monkey 8 hr following 6,000 r. X 85. (E.) A severe perivascular leucocytic 
infiltration in the parietal cortex 6 hr following 4,500 r. X 85. 

accelerated penetration of Na-* into the brain and cerebrospinal fluid in 
the radiated animals, even though histologic changes were minimal even 
at 1,500 r. 



EFFECTS ON BLOOD-BRAIN BARRIER, CEREBRAL BLOOD \ ESSELS 417 







Fig. 4. This figure demonstrates delayed white matter degeneration (arrows) seen 
in the internal capsule of a monkey given 1.500 r to the head and sacrificed 8 months 
after x-irradiation. These regions showed areas of demyelination and neuroglial scar 
formation. Clajal-gold sublimate impregnation. X 80. 



One is left to wonder whether our methods of determining the effects of 
lower x-irradiation doses on the aduh brain ha\-e been, to this time, sharp 
enouoh to detect the more subtle changes which may be taking place. The 
axiom that the brain is relati\ely resistant to radiation mi^ht be questioned 
purely on the lack of sensitix e methodolooy. When Rose i 1958) is able to 
detect vascular permeability changes iitilizino- only several hundred roent- 
gens, and Ganolofl" and Haley 1960) show that only 200 to 400 r are re- 
quired to alter spontaneous and exoked electrical actixity in certain parts of 
the brain, it is time to reconsider our opinions of the radiosensiti\ity of the 



418 CLEMENTE AND RICHARDSON 

central nervous system. In light of the fact that morphologic changes in 
cells and fibers at these low dose levels are unobservable, perhaps the func- 
tional alterations observed are the result of an altered neuronal milieu or 
changes in the ionic medium of neuronal pools which may be transient at 
lower doses or may result in neuronal damage or neuron death at somewhat 
higher doses. It seems evident that at extremely high doses of radiation, 
vessels, glia, and neurons all sufTer damage. There may be a level of radia- 
tion, however, at which the neuronal damage is secondarily brought on by 
damage to the functional integrity of the capillary system. 



Radiation Effects on Cerebral Vessels 

Doses of ionizing radiation to the brain which have been reported capable 
of producing radiation lesions have, in virtually every instance, involved 
not only nerve cells and fibers, but also the vascular system. The vascular 
reaction to radiation in the brain is characterized by generalized vasculitis, 
brain edema, and vascular fragility I Lyman ct al., 1933, 1952; DavidofT 
et al, 1938; Russell et al, 1949; Colwcll and Gladstone, 1937; Haymaker 
et al, 1958). Similar vascular reactions following irradiation have been 
described in skin ( Wolback, 1909, 1925; MacKee, 1938; Miescher, 1930; 
Snider, 1948), lung (Englestad, 1934; Warren and Spencer, 1940), and 
kidney (Warren, 1936). 

Observations on the fate of capillaries in radiated skin and in developing 
granulation tissue reveal that endothelial protoplasmic buds retract and be- 
gin to become obliterated at about 400 r, even though inhibition of granula- 
tion tissue does not occur until 2,000 r is administered. Under these circum- 
stances there is swelling of the endothelium, producing first a narrowing of 
the lumen and then a dilitation which results in a primary radiation ery- 
thema. Edema ensues, apparently being dependent on the reactivity of the 
capillary endothelium and of the surrounding parenchyma (Van den 
Brenk, 1959; Borak, 1942a,b,c). Following the edema, diapedesis of cellular 
elements through the vessel wall occurs, involving lymphocytes, red blood 
cells, and polymorphonuclear leucocytes. This results in the commonly ob- 
served inflammatory response of the perivascular tissue to radiation. 

One of the reported effects of ionizing radiation on cells is the intracellular 
disintegration of large protein molecules into those of smaller size. This re- 
sults in an increase in osmotic pressure leading to imbibition of fluid by the 
cell (Heilbrunn and Mazia, 1936; Borak, 1942a). Endothelial cells in the 
brain prove no exception. An increase in basophilia and endothelial cell 
swelling were among the first reactions observed by Clemente et al ( 1959, 
1960) in cerebral capillaries of neonatal rats subjected to 500 r doses of 



EFFECTS ON BLOOD-BRAIN BARRIER, CEREBRAL BLOOD VESSELS 419 

x-rays. With his:her doses, these vascular reactions were followed by petechial 
hemorrhatres at the capillary le\el and polymorphonuclear perivasculitis. 

A pattern of cerebral vascular damage followins,- radiation could be ob- 
served. The earliest and commonest change with the lowest radiation thresh- 
old was a simple swelling of the endothelial cell with an increase in nuclear 
basophilia. With a longer survival time this process either was arrested and 
re\ersible (with lower radiation dosages) or progressed to peri\asculitis or 
the development of petechial hemorrhages (with higher radiation dosages, 
Clemente ct al., 1960). Russell ft al. i 1949) described similar findings in the 
brains of rabbits. 

Radiation damage in larger sized arteries and \eins becomes manifested in 
the pathologic changes observable in vessel walls. The inflammatory radia- 
tion reaction in the vascular tunics of the brain seems indistinguishable from 
arteritis or phlebitis obser\ed during inflammation in other tissues. Incom- 
petency of the vessel walls leads to plasma leakage and cellular migration. 
These degenerative and subsequent reparative phenomena in cerebral vessels 
ot radiated brain contribute to narrowing of the lumen and at times to com- 
plete occlusion of the vessel with apparent cessation of blood flow. Charac- 
teristically obser\ed is intimal fibrosis and hyalin degeneration of fibrous 
and muscle tissue. Ot prime importance is the fact that a radiation dose 
capable of producing exudation and leucocytic emigration through the en- 
dothelium of capillaries is not especially sufficient to produce a generalized 
inflammatory reaction in the walls of larger arteries and veins i Borak, 
1942a,b,c). A considerable increase in dose is necessary to produce the gen- 
eralized vasculitis often described. We wish to stress the fact that low doses 
to the brain i under 1,000 r) are capable of interfering with the competency 
ol the cerebral capillary system and that this may eventually be manifested by 
localized or more widespread efTects on the electrical activity of neuronal 
pools, even though histopathologic efTects have not often been detected at 
these dosage levels. 

The delayed white matter necrosis obser\ed (Fig. 4) by Clemente and 
Hoist ( 1954 I in monkeys who.se heads had been radiated with 1..500 r ( 4 to 
8 months before sacrifice ) may have been a chronic manifestation of nerve 
fiber pathology due to small blood vessel damage. This .seems especially 
likely since the areas of degeneration were close to small \essels whose walls 
revealed intimal thickening. Animals in this group which were administered 
trypan blue just before sacrifice showed a blue staining of the brain in the 
degenerated areas only, another indication of a localized incompetent \ascu- 
lature at the lesion sites. 

The phenomenon of delayed cerebral necrosis due to radiation is difficult, 
if not impossible, to explain except by implication of \ascular pathology. 
The necrotic areas are limited to the regions irradiated and the immediate 



420 CLEMENTE AND RICHARDSON 

surrounding zones (Courville and Myers, 1958). For the most part, authors 
reporting this process describe vascular changes of such a nature as to ac- 
count for the necrosis either on a hemorrhagic or ischemic basis (Marburg 
et al., 1945; Pennybacker and Russell, 1948). Analysis of vascular reactions 
to irradiation in other tissues revealed that following irradiation it takes 
about a month to produce an ischemic ulcer in the skin(Borak, 1942a). Once 
a segment of skin has been irradiated, it continues to be more susceptible to 
noxious influences such as heat, cold, infection and trauma. Dynes and 
Smedal (1960) report a case of radiation necrosis precipitated by trauma, 
and other authors suspect a progressive deterioration of blood vessels follow- 
ing high doses of radiation (Russell et al., 1949; Berg and Lindgren, 1957; 
Bailey f^ al, 1957). 

More unique experiments have been reported in which brain destruction 
does not seem to be a direct result of vascular damage. Malis et al. (1957) 
reported the production of laminar lesions in the cerebral cortex with a 
minimum of 15,000 rads peak dose. This is 2 to 3 times the minimum dose 
necessary to produce a lesion when a large volume of brain is irradiated with 
a uniform dose. Even under these conditions, capillaries appeared to dilate 
early, and occasionally extravasation of blood elements occurred. Older 
"line lesions" showed thickening of capillaries (J. E. Rose et al., 1960). 
Zeman et al. (1959) irradiated mouse brains with beams of deuterons of 
varying diameters. The beam diameters ranged from 1 mm to 25 jx. They 
found that 15,000 rad were below a minimal effective dose for a 1 mm 
diameter beam, 75,000 rad were below the minimal efTective dose for a. 75 fx 
beam and 5.5 X 10' rad were not minimally effective with a 25 /x beam. 
With the smaller beams, little or no involvement of the vasculature occurred, 
presumably because little or no vasculature was in the field; hence, under 
these conditions, the production of a lesion seemed dependent on the "direct 
killing" of brain parenchyma. 

In 28-day-old rats, the effects of alpha particle radiation on the vascula- 
ture and parenchyma of the central nervous system were studied. The radia- 
tion was delivered to the lower bulbar or upper cervical spinal cord region 
from the 184 in. cyclotron at Berkeley, California. The beam was directed 
through 1X5 mm slit aperture and delivered at approximately 2,000 rad 
per min with doses of 10,000 or 12,500 rad. In other animals, the beam was 
directed through a 3 X 4 mm aperture over the cerebellum. Animals were 
sacrificed at varying periods up to 4^2 months after irradiation. Often a 
rather large cyst developed in the central nervous system at the radiation 
site (Fig. 5A), but perhaps the most consistent observation was thickening, 
hyalinization, and often occlusion of the blood vessels. Both surface vessels 
(Figs. 5B and 5C) and deep vessels (Figs. 5D-F) were affected. In certain 
areas (Figs. 6A and 6B), virtually all smaller vessels were hyalinized and 


















Fig. 5. These 6 pictures demonstrate histologic material taken from the brain ot 
rats subjected to 10.000 rad alpha particle radiation delivered through a slit aperture 
from the 184 in. cyclotron at Berkeley. California. Van Gieson's \ascular stain. (A.) 
Low power view of cervical spinal cord (level C-1 ) 2 months after radiation. Note 
the pronounced cyst formed in the dorsal funiculi. X 27. (B and C.) Examples of 
perivascular infiltration, vascular lumen thickening, and almost complete occlusion of 
the left (B) and right (C) dorsal descending spinal arteries (surface vessels) taken 
froin the same level as shown in Fig. 5A. X 310. (D, E, and F.) Three examples of 
perivascular infiltration, thickening, and hyalinization of deeply situated vessels in 
the brain. Rat sacrificed 3 months after radiation. These \essels were situated in the 
nucleus gracilis. (D) X 250: medullary reticular formation, (E) X 480; and central 
medulla at the level of the pyramids. (F) X 480. 

421 



422 



CLEMENTE AND RICHARDSON 







• ^ 







^*.,^^' 










■i€ 



#*'. 



'♦ ;. 



.*^ 



w* . '■ • 



' t . ' 










Fig. 6. (A.) Medium power view of the nucleus of the spinal tract of the trigeminal 
nerve in same rat as 5D,E,F, sacrificed 3 months after irradiation with 10,000 rad 
alpha particles. Note the widespread vascular hyalinization (arrows) and the absence 
of normal-looking neuron cell bodies. Van Gieson's stain. X 360. (B.) Low power 
view of 3 vessels in the white matter in the occipital corte.x of a cat 6 weeks after 
irradiation with 10,000 rad alpha particles. X 250 (C and D.) Sections at the mid- 
pontile le\-el in rat gi\en 12,500 rad alpha particles and sacrificed 10 days after radia- 
tion. Note the frank hemorrhages (C) X 360, and the perivascular globules (arrows) 
described by Brightman (1959) (D) X 560. 



EFFECTS OX BLOOD-BRAIN BARRIER, CEREBRAL BLOOD VESSELS 423 

partially or completely occluded. In these reoions, few. if any, normal- 
looking nemons could be found. In another animal, sacrificed 10 days after 
the beam was directed at the brain stem, there were frank hemorrhagic areas 
in the midbrain ( Fig. 6C ) and peri\ascular globules (Fig. 6D I in the brain 
stem, similar to those reported by Brightman I 1959). 

Vascular and parenchymal damage was also observed in the cerebellum 
following alpha particle radiation. This was characterized by spongy loss of 
cerebellar cortical tissue, thickening of the meningeal and inxaginating 
vessels, and \acuolization of the Purkinje cell layer ( Figs. 7A-D i . 

In these higher radiation dose experiments, it is difficult to say that the 
entire neuropathology observed is the result of an altered circulatory system. 
On the other hand, it is impossible to believe that the neurons in a portion 
of the neuraxis with such badly damaged vessels could react physiologically. 



Summary 

In this communication we wished to slant our attentions to the reactivity 
of the vasculature in the brain and spinal cord to ionizing radiations. Studies 
dealing with the influence of x-ravs on the blood-brain barrier were re- 
viewed, and pathologic changes in central nervous system blood \essels due 
to x-rays and to alpha particle radiation were described. 

It is felt that x-iadiation dosages under 1.000 r administered to the brains 
of adult animals fail to reveal marked histopathologic findings, even though 
these ha\e been observed following this radiation dosage in newborn animals. 
Nevertheless, the effect of these relati\-ely low doses may become manifested 
in nein^al dysfunction through changes in the surrounding medium of 
neuronal pools. Thus, we feel that one of the effects of low dosage radiation 
is an increase in the permeability of cerebral \essels. 

With higher x-ray doses (4,500 or 6,000 r) and with alpha particle radia- 
tion (10,000 rad) to the central nervous system, histopathologic findings 
have been obser\ed not only in the cerebral vasculatme. but also in neurons 
and neuroglia. Vascular damage was evident by thickening and hyalinization 
of the vessel walls. Perivascular infiltration of leucocytes and complete occlu- 
sion of larger and smaller vessels were often observed. Neuronal damage was 
evident by morphologic changes in the cytoplasm and nuclei of remaining 
cells and by spongy loss of neuronal elements in the areas irradiated. Neiu'ons 
showing \acuolization and ghost cells were frequently observed. Although at 
these higher dosages it was difficult to maintain that the neuronal pathology 
was only the direct result of altered circulation, it is felt that vascular 
pathology cannot be imderestimated in an analysis of the response of the 
central nervous svstem to ionizing radiations. 



424 



CLEMENTE AND RICHARDSON 






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Vh,. 7. Set lions of cerebellum in idls administered 12,5UU rad alpha particles. 
(A.) Rat sacrificed 7'/; weeks after radiation. Note the spongy degeneration of the 
cerebellar cortex. X 63. (B.) Higher power view of cerebellar surface pictured in 7A, 
showing degenerative vacuolization, miningeal thickening, and vascular hyalinization 
(arrows). X 360. (C.) Another rat 10 weeks after radiation. Note the greatly thickened 
surface cerebellar vessels. X 175. (D.) High power view of cerebellar cortex in rat 
described in 7C. Note the extensive Purkinje cell loss just under the cerebellar molecu- 
lar layer. One pyknotic cell (arrow) still remains while a few nuclei of other vacuolized 
Purkinje cells can also be seen. X 500. 



EFFECTS 0\ BLOOD-BRAIN BARRIER, CEREBRAL BLOOD VESSELS 425 

Referen'ces 

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426 CLEMENTE AND RICHARDSON 

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EFFECTS ON BLOOD-BRAIN BARRIER, CEREBRAL BLOOD VESSELS 427 

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Chemical and Enzymatic Changes in Nerve 
Cells Irradiated with High Energy Deuteron 

Microbeams* 

Wolfgang Zeman, Howard J. CIurtis, Dante G. Scari'elli, 
AND Ruth Kleinfeld 

Indiana University Medical School, Indianapolis. Indiana. 

Brookhaven National Laboratory. Upton. New York, and 

Ohio State University, Columbus. Ohio 



In previous studies on the mouse brain, it was found that high energy 
deuteron microbeams with a diameter of approximately 25 and 75 ^ will 
produce nerve cell necrosis without any definite pennanent damage to inter- 
stitial elements (Zeman ct al, 1959j. The first stages of this process can be 
observed in histologic preparations at approximately 4 days after a surface 
dose of about 400,000 rad delivered by a beam 0.025 mm in diameter. After 
24 days, a complete loss of nerve cells has occurred, located strictly within 
the area of the beam path. In other words, the lesion forms a cylinder, the 
dimensions of which are determined by the reducing apertures and by the 
depth range of the particles (Fig. 1). Nerve fibers, myelin sheaths, and 
vessels do not appear altered. 

These findings are in contrast to lesions resulting from wider beams. With 
beams of 1 mm diameter, it is impossible to produce only a selective nerve 
cell necrosis within the beam path. With a surface dose of up to 4.800 rad, 
nothing happens to the irradiated cortical neurons. With higher doses, 
there is focal tissue necrosis resulting in small cystic lesions, which occur 
predominantly at the end of the depth range of the deuterons (Fig. 2). To 
destroy all ner\e cells within the path of a 1 mm beam, about 14.000 rad is 
required, but this dose destroys all the other tissue elements as well. 

Since there is apparently no dosage using beams of 1 mm in diameter 
which would selecti\ely destroy ner\e cells within the path of the beam with- 
out also destroying the interstitial elements, microbeam irradiations seem to 
aflford a unique opportunity to study one of the two different dose-dependent 

* This study was supported by a U.S. Public Health Ser\ice Grant and was per- 
formed in part at Brookhaven National Laboratory under the auspices of the U.S. 
.'Atomic Energy Commission. 

429 



430 



ZEMAN, CURTIS, SCARPELLI, AND KLEINFELD 









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Fig. 1. U.UJj nun Ix-ani ; 'Jljniii i^d. 17 days sui\i\>il. \ imi.iI > oitex. The beam 
path is characterized by the loss of nerve cell bodies which are frequently represented 
by holes. Note the apparently normal capillary traversing the lesion. PAS-Gallocyanin. 



CHEMICAL CHAxNGES IN IRRADIATED NERVE CELLS 431 




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Fig. 2. 1 mm beam: Ib.bUU rad. 7 days survival. Hippocampus. Note complete 
tissue necrosis with cavity formation, hemorrhages in molecular layers and beginning 
formation of compound granular corpuscles. PTAH. 

pathogenic mechanisms of radiation damage of the brain tissue obser\ed by 
Schiimmeheder (1959). namely direct radiogenic neive cell necrosis. 

Preliminary experiments were designed to elucidate some chemical events 
incident to this necrosis. 

Methods 

Carworth CF, female mice. 6 to 7 weeks old, weighing 25 to 35 gm, were 
irradiated with different doses and beams and were sacrificed after 1, 2, 3, 
4. 5. and 7 days. The animals w^ere anesthetized and the brains were fixed 
by in situ perfusion with Heidenhain's Susa fluid. After being embedded in 
paraffin, the brains were cut in serial sections, even- second section being 



432 ZEMAN, CURTIS, SCARPELLI, AND KLEINFELD 

stained with PAS-gallocyanin. After the radiation-induced lesions has been 
identified on these slides, the alternate sections were stained for cytoplasmic 
proteins with Fast Green at pH2, for cytoplasmic RNA with Azure B at pH 
4, and for nuclear DNA according to Feulgen's technique after digestion 
with RN-ase. 

On these preparations, the following observations could be made. Depend- 
ing on dose, the development of nerve cell necrosis begins at 2 to 5 days 
after irradiation; higher doses usually resulting in a shorter latency. Using 
beams of 75 /x and less, and doses of 100,000 rad and more, most nerve 
cells within the beam path show a synchronous pattern of necrosis, while 
wider beams and doses of less than 100,000 rad produce nerve cell necrosis 
in a random distribution, i.e., some neurons are in a state of complete disinte- 
gration, while others are still unaltered. The first stage of this radiation- 
induced nerve cell necrosis consists of disintegration of the cytoplasm. About 
the same time, the cytoplasmic DNA disintegrates. The nerve cell nuclei, 
however, remain unaltered and appear naked. It is difficult to estimate how 
long the nuclei rest in this state. They eventually become pyknotic, disinte- 
grate, and disappear completely. It is not clear which cellular elements 
finally phagocytize the debris from the necrotic neurons ; however, it appears 
that the nerve cell necrosis takes place without any demonstrable evidence 
of microgliocytic proliferation or activation. 

From these obseivations, it was assumed that radiation-induced nerve cell 
necrosis might conceivably be an autolytic process generated by the activa- 
tion of proteolytic enzymes within the irradiated ner\e cells. To test this 
hypothesis, the following studies were undertaken. 

Fourteen mice received multiple beam irradiations over the visual cortex 
and the cerebellum. Each animal of the 1st group received 4 irradiations 
with 0.250 mm beams at 30,000, 60,000, 120,000, and 240,000 rad. Those 
of the 2nd group were given 8 irradiations with 0.075 mm beams at 90,000, 
180,000, 270,000. and 540,000 rad. These doses were measured in the ioniza- 
tion chamber, thus indicating relative, but not necessarily absolute, tissue 
dosages. The beams were directed to the visual cortex and the cerebellum by 
means of telescopic focusing. They were arranged in a frontal plane, spaced 
60 mils from center to center. The animals were sacrificed by neck disloca- 
tion at 4, 7, 10, and 30 hours after irradiation. 

The brain was immediately removed and fixed in formol-calcium at 4° C. 
After 24 hours fixation, the brains were cut in serial sections at 15 ^ on a 
standard freezing microtome. The sections were incubated in a medium 
prepared after Holt I 1958) at pH 5 for demonstration of esterases. To block 
carboxylic esterases of the acetylcholinesterase, pseudocholinesterase, and 
lipase type, the medium contained a 10"" M concentration of diisopropyl- 
fluorophosphate. Thus, only A-type cathepsins were believed to be stained. 



CHEMICAL CHANGES IN IRRADIATED NER\ E CELLS 433 

The frozen sections were counter-stained with a variety of methods, such as 
Oil Red "0"\ Nuclear Fast Red. or azocarmine, or were impregnated with 
siKer carbonate, and moimted. 

Results 

Althou2,h extreme care was exercised in processing the sections, radiogenic 
lesions were discovered and adequately demonstrated in only 5 brains. A 
variety of factors, such as the minute size of the lesions, the tremendous 
number of sections i over 2.500 i. and possible errors in the placing of the 
lesions, probably accoimts for the high rate of failure. Nonetheless, the 
material available for studv yielded interesting results. 

Animal No. 4 was subjected to 4 irradiations with 0.250 mm beams and 
was sacrificed 5 horns later. Only the lesions produced with 120.000 and 
240.000 rad. respectively, were found histologically. The irradiated nerve 
cells appeared shrunken and hypcrchromatic in silver-carbonate impregna- 
tions. No abnormal enzyme activity was noted. 

Animal No. 5 received 4 irradiations with 0.250 mm beams and was per- 
mitted to survive 10 hours. The lesions produced with 120,000 and 240,000 
rad. respectively, were foimd in the visual cortex. The nerve cells were 
shrunken and hypcrchromatic. They did not show anv enzyme activity. On 
the other hand, the vascular endothelial cells exhibited considerable activity 
in the form of densely packed blue granules in the cytoplasm. 

Animal No. 6 received 4 irradiations with 0.250 mm beams and was sacri- 
ficed after .31 hoius. Only the lesions in the visual cortex, produced with 
60.000 and 240.000 rad. respectively, were foimd. The nerve cells were 
shrunken, and a few of them contained blue granules in the cytoplasm. In the 
dentate ligament of the Amnion's horn, the enzyme appeared to be located 
predominantly in endothelial and proliferating microglia cells and in astro- 
cytes. The nerve fibers of the callosal radiation were imaltered. while those 
ot the cortical radiation exhibited retraction bulbs. 

Animal No. 8 received 8 irradiations with 0.075 mm beams and was sacri- 
ficed after 10 hours. Only one lesion in the visual cortex, produced with 
540,000 rad. was foimd. .\11 nerve cells in the beam path exhibited consider- 
able enzyme activity in their cytoplasm. The nuclei were shrunken and 
stained a deep red with azocarmine. 

Animal No. 9 received 8 irradiations with 0.075 mm beams and was killed 
30 hours later. Six lesions were identified, produced with 180.000, 270,000, 
and 540,000 rad. respectively. By sheer chance, in one section of the cere- 
bellum. 3 lesions were cut on the same plane i Fig. 3 ) . In these lesions, the 
enzvTOe activity was almost e.xclusively restricted to the Purkinje and granu- 
lar cell lavers. In contrast, the white matter showed no activity at all, and 



434 



ZEMAN, CURTIS, SCARPELLI, AND KLEINFELD 




Fig. 3. 0.075 mm beam: (from left to right) 540,000 rad, (site of lesion is torn), 
270,000 rad, and 180,000 rad. 30 hours survival. Cerebellum. Note that the path of 
the beam is outlined by the dark staining blue indigo granules developed by the ac- 
tivity of the cathepsin-like enzymes. Frozen section. Holt's technique for esterases, 
blocking with DFP, counter-stained with Oil Red "O". 



the molecular layer contained only a few spots of activity (Fig. 4) . Much of 
the latter appeared to be in capillary endothelial cells and astrocytes, but 
some of the superficial stellate cells also exhibited blue granules in their 
cytoplasm. 

Owing to the thickness of the sections, it was extremely difficult to prop- 
erly localize the site of enzyme activity in the Purkinje and granular cell 
layers. In some instances, however, chance permitted us to make more defi- 
nite statements. In Fig. 5, one can readily discern the emergence of com- 
pound granular corpuscles, loaded with blue indigo granules from a capillary 
wall. Figure 6, on the other hand, shows 2 Purkinje cells, irradiated with 
540,000 rad, having a deep blue cytoplasm. It can also be noted that their 
baskets are apparently unaltered. Some of the adjacent granule cells are 
similarly affected. Figure 7 depicts the granule cell layer of the hippocampus 
where it was hit by a beam of 180,000 rad. Here, the cytoplasm of many 
neurons is stained a deep blue. 



CHEMICAL CHANGES L\ IRRADIATED NERVE CELLS 



435 




Fig. 4. 0.075 mm beam: 180.000 rad. 30 hours sur\-ival. Cerebellum. Note the 
degree of enzyme activity in the different layers, (M) molecular layer, (P) Purkinje 
cell layer, (G) granule cell layer, and ( W ) white matter. Frozen section. Holt's tech- 
nique for esterases, blocking with DFP. counterstained with Oil Red "O". 

Discussion 

One cannot overlook the marked difTerence between the activity of A-type 
cathepsins as elicited by small and lar2,e diameter beam irradiations. While 
the path of 0.075 mm beams is sharply outlined by the blue indi«o formed by 
the enzymes, the 0.250 mm beam path, after an almost identical dose and 
survival time, displays only sporadic evidence of increased enzyme activity. 
Furthermore, in the lesions produced by the small beams, the enzyme activity 
is found just as often in the nerve cell cytoplasm as in the interstitial elements, 



436 ZEMAN, CURTIS, SCARPELLI, AND KLEINFELD 











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Fig. 5. U.U75 mm beam: 540, (JUO rad. 3U hours sur\i\al. Ctrcbellum. Capillary 
with emerging compound granular cells. Frozen section. Holt's technique for esterases, 
blocking with DFP, followed by siKer-carbonate impregnation. 

while in the lesions produced with 0.250 mm beams, the enzymes are pre- 
dominately active within the interstitial cells. The degree of enzyme activity 
is in direct proportion to the density of the nerve cell popidation. Thus, in 
white matter, and to a lesser extent in the molecular layers, radiation-induced 
activity of cathepsin-like enzymes is practically nil. 

The difference between the enzyme activity as elicited by beams of two 
different sizes might be somehow related to dose rates. All lesions were pro- 
duced with a high dose rate of 63,000 rad per sec. While such a dose rate 
permits the enzymatic changes to develop with small beams, it appears to 
introduce physico-chemical changes in the path of wider beams which hinder 
the activation of cathepsin-like enzymes. Support for this assumption is 
derived from previous studies in which one hemisphere of the mouse brain 

Fig. 6. 0.075 mm beam: 540,000 rad. 30 hours survival. Cerebellum. Note 2 Pur- 
kinje cells with darkly staining cytoplasm. Some of the granule cells are similarly 
affected, but this is difficult to appreciate on the photograph. Frozen section, Holt's 
technique for esterases, blocking with DFP, followed by silver-carbonate impregnation. 

Fig. 7. 0.075 mm beam: 180,000 rad. 30 hours survival. Hippocampus. Cathepsin 
activity in the cytoplasm of the granule cc41s. Holt's technique for esterases, blocking 
with DFP, counter-stained with azocarmine. 



CHEMICAL CHANGES IN IRRADIATED NERVE CELLS 



437 




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438 ZEMAN, CURTIS, SCARPELLI, AND KLEINFELD 

received 8,000 rad with 5 mm beams at a rate of 1,600 rad per sec. At 72 
hours after irradiation, numerous nerve cells showed cathepsin activity in the 
cytoplasm. The active nerve cells were randomly distributed. 

The occurrence of cathepsin-like enzymes in the nerve cell cytoplasm after 
irradiation is difficult to explain. Numerous controlled studies have shown 
that, except for certain groups of neurons located in the hypothalamus and 
the motor and reticular nuclei of the brain stem, the nerve cell bodies do not 
contain cathepsin-like enzymes as demonstrated with the technique used in 
this study. This would mean that ionizing- radiation can either activate the 
precursor of such enzymes or hberate them from the lysosomes in which 
they are locked up, according to de Duve (1959). That such submicroscopic 
particles are abundantly present in nerve cell cytoplasm has been shown by 
Scarpelli and Zeman (1960) in electron micrographs. Further studies to test 
the latter hypothesis are underway. 

The occurrence of radiation-induced proteolytic activity in nerve cells 
might help to explain radiation-induced central nervous system excitation 
consistently being observed and described by Russian investigators (Stahl 
1959). Evidence for the causal correlation of cellular excitation and pro- 
teolytic activity has been amply provided by the work of Nassonow and his 
school (discussed by Troschin, 1958). It could also be entertained that some 
of the early clinical manifestations of radiation, such as shock and radiation 
sickness, might be related to a release of proteolytic enzymes into the circu- 
lating blood. 

In conclusion, it can be said that the present studies, although preliminary 
and incomplete, might yield new knowledge in regard to certain phenomena 
of radiation biology. 

Summary 

It has been shown that the direct radiation injury of nerve cells is asso- 
ciated with the occurrence of proteolytic enzymes within the nerve cell 
cytoplasm. 



CHEMICAL CHANGES L\ IRRADIATED NERVE CELLS 439 

References 

de Du\e. C. 1959. Lysosomes a new group of cytoplasmic particles. In "Subcellular 

Particles" (T. Hayashi. ed.). Ronald Press, New York. 
Holt, J. S. 1958. Quoted from Pcarse A. G. E. 1960. "Histochemistry." Little, Brown, 

Boston. 
Scarpelli, D. G., and Zeman, W. 1960. L'npublished data (locally presented). 
Schiimmelfeder. N. 1959. Der \'erlauf der experimentellen Strahlcnschadigung des 

Hirngewebes. Verhandl. deut. Ges. Pathol. 42, 244-250. 
Stahl. W. R. 1959. A Re\iew of Soviet Research on the central nerxous system effects 

of ionizing radiations. /. Nervous Mental Disease 129, 511-529. 
Troschin, A. S. 1958. "Das Problem der Zellpermeabilitat." Fischer, Jena. 
Zeman, W.. Curtis. H. J., Gebhard. E. L.. and Haymaker, W. 1959. Tolerance of 

mouse-brain tissue to high-energy deuterons. Science 130, 1760-1761. 



Tolerance of Central Nervous System 
Structures in Man to Thermal Neutrons* 

L. E. Farr, W. G. Calvo, Y. L. Yamamoto, E. E. Stickley, 
W. Haymaker. t and S. W. Lippincott 

Brookhaven National Laboratory, 
Upton, Neiv York 



During the past several years at Brookhaven National Laboratory, the 
experimental procedure of neutron capture therapy of intracranial neo- 
plasms has been investioated utilizing a nuclear reactor as a neutron source 
I Farr ct at., 1954-1960). In neutron capture therapy there are two main 
components to the therapeutic system : the capture or target atom and the 
thermal neutron or triggering component. As a result of thermal neutron 
capture, an atomic transformation occius with release of energy. When the 
target atom is boron- 10. the thermal neutron capture triggers an immediate 
disintegration of the B'" into an energetic lithium-7 atom and an energetic 
alpha particle with a modest gamma ray emission, totaling an energy release 
of 2.8 Me\'. The restricted distribution of the energy release to a volimie of 
one average cell proxides a selectixity of action that, in principle, will aflfect 
one cell suitably primed and loaded, while the adjacent cell is totally un- 
scathed. It is of importance in the development of a new therapeutic 
modality to determine not only the efl'ect on the disease process, but also the 
possible effects on residual normal cell structiues. Since large mnnbers of 
thermal neutrons must penetrate tissue within a few minutes at most, the 
tolerance of the central nervous system to thermal neutrons becomes a matter 
of great practical, as well as theoretical, interest. Fortunately, the conditions 
of therapy permit a study of neutron effects to be made relati\ely inde- 
pendent of the B^" captiue reaction, since the distribution of the two com- 
ponents of the reaction differ widely at the time of exposure. 

This presentation deals with observations made on patients so treated, 
with one patient receiving only thermal neutrons. In addition to the clinical 
obsenations, a combined topographic and histopathologic surxey was made 
of the irradiated brains. 

* This research was supported by the U. S. .\toniic Energy Commission. 
t Research collaborator from the Armed Forces Institute of Pathology, Washing- 
ton. D. C. Present .Address: N.^S.^ .^mes Research Center. Moftett Fifld. California. 

441 



442 FARR ET AL. 

The underlying purpose has been to determine whether the reaction was 
capable of destroying neoplastic cells and whether surrounding and distant 
structures were affected by the procedure. To make this evaluation, a com- 
parison series of cases was collected to determine the spontaneous alterations 
which occur in untreated cases, e.g., the amount of necrosis occurring in the 
development of a neoplasm of the glioblastoma multiforme type, the most 
commonly investigated neoplasm in this study. In addition, forms of irradia- 
tion other than neutron capture therapy, such as that occurring in x-ir- 
radiation, have been considered for comparative purposes. Thus, in the 
nonirradiated and irradiated brain, the whole brain was embedded in 
celloidin, with whole brain sections being cut in coronal, horizontal, or 
sagittal planes at 25 ^ and mounted on slides for low power survey. In addi- 
tion, representative sections were cut at 7 /x and mounted with thin cover 
slips for detailed microscopic examination. A variety of special stains de- 
signed for studying the cellular details of the central nervous system with 
the nerve tracts weie used for detailed obsei'vations. 

The major series comprised 16 patients to whom neutron capture therapy 
was given in 1 to 4 exposures with, in the various patients, a total thermal 
neutron exposure ranging from 0.44 X 10^" to 6.31 X 10^' per square cm 
at the skin surface. An additional patient was treated at the new medical 
research reactor in a single exposure with a total thermal neutron penetra- 
tion of 1.73 X 10'" per square cm at the cortical surface. Studies are still in 
progress on another patient who, in a similar fashion, received over 10'^ 
neutrons per square cm on the cortical surface. In contrast to these cases 
with neutron capture therapy, one patient received thermal neutrons only, 
and this data provides comparison material. 

Neutron capture therapy is accompanied by radiation other than that 
specifically provoked. Thus, it becomes necessary to establish the levels of 
these ancillary radiations and to evaluate whether they contribute to or 
detract from the clinical picture. The several contaminating radiations 
include ( 1 ) gamma radiations from the reactor core and gamma rays 
induced when neutrons are lost through capture in the shielding and reflect- 
ing materials, (2) the remaining small fraction of the emergent neutrons 
which retain energies in the kilovolt range which may possibly cause tissue 
damage in their own right, and (3) the effects of the passage of the great 
quantities of slow neutrons through tissue, wherein, through thermal neutron 
capture by hydrogen and carbon, there is produced appreciable induced 
gamma emission; and by nitrogen, energetic protons. The first two factors 
are approached chiefly as engineering design problems; the third is the sub- 
ject of this essay. 

Neutrons are elementary nuclear particles which have essentially unit 
mass, about the size of an hydrogen nucleus or proton, but no electrical 



HUMAN TOLERANCE TO THERMAL NEUTRONS 443 

charge. We know that neutrons of higli energy, from 0.1 or 0.2 Mev and up, 
do have an extensive damaging eflfect on tissue. These neutrons are able to 
disrupt molecular relationships and otherwise cause disturbance of the 
normal life pattern. These efTects arise largely from the mechanical impact 
of fast neutrons on atomic nuclei. It is the lack of such high levels of kinetic 
energy which make the slow neutron innocuous in this respect. 

When neutrons of thermal energy, namely 0.025 ev, penetrate into tissue, 
they do not directly cause damage by their passage ; rather, they pervade the 
tissue as a gas pervades whatever space it is released into. However, with 
increasing penetration their numbers significantly and predictably decrease 
as they travel from the surface through solid tissues. The pattern of attenua- 
tion is shown in Diagram I. Any effects may be expected to result only from 
capture with atomic transformations, radioactivity resulting therefrom, or 
both. 

Radiations coming from the induction of acti\ity in tissue components 
ha\e to be recognized in assaying the total efTects of neutron exposure of 
tissue. Chief among these reactions are the capture of slow neutrons by 
hydrogen, resulting in an immediate gamma ray emission at over 2 Mev, the 
capture of slow neutrons by nitrogen, giving an instantaneous proton of 
energy 0.6 Mev, and the capture of slow neutrons by carbon, yielding gamma 
rays of up to 8 Mev. Other constituents in tissue, particularly sodium and 
chlorine, may contribute readily measurable additional radiation. 

In neutron capture therapy, a much greater intensity of reaction is in- 
duced in the treated tissue by providing thermal neutrons in large numbers 
at such a time after the target atom injection as we empirically have shown 
to be efTccti\e in experimental tumor destruction and sparing of adjacent 
normal tissues. The mechanisms of the cytocidal effect are under intensive 
study, as no explanation is conveniently provided by chemical analyses of 
target atom species for the differently affected tissues. 

In the experimental in\estigation of neutron capture therapy at Brook- 
ha\en, two reactors ha\e been used as sources of neutrons. The work was 
begun at the large graphite research reactor when it first reached its full 
operating power in 1951. A part of its biologic shielding was modified to 
provide a hollow cone delivering neutrons through a treatment aperture. 
Subsequent improvements in this arrangement provided a shutter and a 
higher flux of neutrons. Some 40 experimental procedures with patients, 
together with studies on mice, rabbits, and swine at the graphite research 
reactor, gave experience and background for the design and construction of 
the medical research reactor, a reactor entirely oriented toward studies in 
man. The details have been published pre\iously ( Farr, 1959). 

Dosimetry of neutron capture-induced gamma radiations in tissue is not 
a straightforward measurement, since there is no detector which, in a "amma 



444 



FARR ET AL. 




CO E 



jUUO/ LI 




HUMAN TOLERANCE TO THERMAL NEUTRONS 445 

field, is completely sensiti\e only to specific particle radiations resulting from 
the reaction. Consequently, we must rely largely on calculations with such 
experimental measurements and obser\ations as can be appropriately carried 
out to establish the \alidity of our conclusions. Since the particle energy 
released is proportional to the concentration of tarcjet atoms and total 
number of neutrons, our measurements are directed to these ends. The con- 
centration of the neutron captiue target element is assayed by chemical 
means, while the total number of neutrons passing a plane is determined by 
activation of gold foils or wires inserted into the neutron penetrated area. 
Gamma ray levels have been calculated and roughly measured by ordinary 
ionization chambers coxered by neutron filters and, experimentally, by 
chemical dosimeters, glass needles, and solid state ionization devices. Fast 
and intermediate energy neutrons in various regions are cxaluated insofar as 
possible by characteristic energy activation foils and fission chamber tech- 
niques. Measurements gi\en in this paper were derived by one or more of 
these procedures. 

Results 

Efiects on nontimiorous neiuonal structmes were studied in 16 patients 
receiving neutron capture therapy for gliomas and sarcomas, with a 20-40 
minute exposure to thermal neutrons. The material discussed is being 
reported in detail elsewhere Farr et ai, to be published). 

Whole brain sections were prepared from 16 cases in which the total 
surface neutron exposure varied from 0.44 X 10^" to 6.31 X 10^" per 
square cm and in which the total dose of tetraborate and pentoborate salts of 
sodium, ranged from 26-50 mg per kg body weight when calculated as B^'^ 
per dose. Experimental exidence has firmly established a correspondence 
between tissue concentration and total close for immediate distribution. 
Borate salts appear to distribute imilormly in body water, eventually reach- 
ing a single equilibrium concentration. In 3 cases of slioblastoma multiforme 
tumor, necroses appeared to be present in the region of the emergent neutron 
port. In a 4th case of cerebellar angiosarcoma and ependymoma, there may 
have been an irradiation effect, but the presence of a fimgal lesion did not 
lead to a clear-cut evaluation. In a 5th case of sarcoma at the site of 



Di.\GRAM L On the left is shown the thermal neutron approximate iso-flu.x con- 
tours in neutrons per square cm with the maximum intensity exposure from the neu- 
trons cloud at the port of entry. The direction of movement of the neutron fog is 
shown, and the fall off in neutron intensity from the port of entry to the midbrain. 
To the right is a graph indicating effects on thermal neutron distribution in tissue 
with different B'" concentrations. Surprisingly little effect is noted therefrom in these 
phantom studies. 



446 



FARR ET AL. 




Fig. 1A. Patient 5972 r. Whole brain section (myelin stain) showing destruction 
of most of the right temporal lobe in which the tumor was located. The areas not 
affected by the tumor, but receiving the maximimi irradiation, show intact myelin. 



maximum neutron intensity, destructive effects to the neoplastic area occurred. 
In the other cases, no alteration in the neoplasms, attributable to neutron 
capture therapy, could be established. Microscopic studies of nontumorous 
and adjacent neuronal structures in all these cases, carried out with various 
staining techniques, gave no suggestion that damage had been done to the 
nonneoplastic central nervous system structures by the irradiation procedure. 
A typical section is shown in Fig. lA. By one set of assumptions, in which a 
uniform neutron exposure is postulated to permit simple calculation, the 
computed dose from tissue-component capture gammas alone (administered 
to the cerebral cortex) ranged from 78 to 1128 rad in these patients. These 
and other calculations were made by Dr. J. S. Robertson of our department. 
Effects due solely to thermal neutron exposure were studied in a man, aged 
53, with glioblastoma multiforme in the right frontoparietal region, who 
was admitted for neutron capture therapy. He had had operative removal 
of part of the lesion and no subsequent x-ray therapy. Due to a technical 
difficulty not appreciated or known at the moment, the patient did not 
receive the injected sodium pentaborate into the carotid artery; thus, when 



HUMAN TOLERANCE TO THERMAL NEUTRONS 



447 



exposed to thermal neutrons, only the tissue comjX)nents were present for 
interaction. The thermal neutron flux at the skin Icxel was 1.875 X 10'' per 
square cm per sec. The irradiation time was 502 sec. The reeion of the tumor 
and the adjacent cortical siuface were exposed to a total of 4.7 X 10" 
thermal neutrons per scjuare cm. The port was 3.5 in. in diameter. Intrinsic 
gamma irradiation from the reactor over this period amoimted to 40 r. 
Death occurred 55 days after irradiation. 

Microscopic examination of the whole brain sections indicated that the 
residual neoplasm had essentially the same histologic appearance as that 
noted in the original biopsy. Thus, as far as could be ascertained, the 
thermal neutrons had no effect on the neoplastic cells. 

The area of the brain adjacent to. and at \arious le\cls distant from, the 
neoplasm was examined microscopically. In the region at the le\el of the tip 
of the frontal lobe, a stain for myelin Fig. IBi shows that there is no loss 
of this material in this region. In contrast, with the same stain (Fig. 2), at 
the le\el of the genu of the corpus callossum, there is extensive demyeliniza- 
tion, in all probability due to the presence of the adjacent tumor. In Fis. 3 
a large mass of neoplasm is shown with partial demyelinization in the ad- 
jacent area in the right occipital lobe. In contrast, the left occipital lobe 




Fig. IB. Whole brain section of case 8180 r at the le\el of the frontal lobes show- 
intj a normal mvcHn content and architecture. 



448 



FARR ET AL. 




Fig. 2. Whole brain section of case 8180 r at the level of the genu of the corpus 
callossum, showing demyelinization of the right temporal lobe and the insular region 
of the same side in the vicinity of the tumor. The frontal lobe has a normal myelin 
content. 



appears completely normal. In the same section the cerebellum may be seen 
with an entirely normal-appearinsi pattern of myelin. The Nissl substance 
and the neurofibrils show no alteration in a section taken from the cortical 
reo'ion receiving' the maximum neutron exposine f Fitj. 4) . 

Effects on nonneiuonal structures were studied in one patient receiving 
neutron capture therapy with high exposure achie\ed in 200 sec. A man, 
aged 44, who had a left frontal craniotomy for oligodendroblastoma, was 
admitted after x-ray treatment following surgery. The surgical wound was 
infected, and there was a brain abscess. Following treatment for the latter, 
it was observed that there was a recurrent deep lying neoplasm in the left 
frontal lobe. He was given 35 mg B^" per kg body weight in the antecubital 
vein. At 31 minutes after the boron infusion, the left frontal area was irradi- 
ated for 200 sec with 1.73 X 10'' thermal neutrons per sc|uare cm at the 
cortical level. Death occurred 30 days after neutron capture therapy. 

Figure 5 shows the whole brain as removed at autopsy. The defect in the 
frontal lobe indicates the area of original operative interference and the site 
of irradiation with thermal neutrons. Fioiue 6 is a coronal section 3 cm from 



HUMAN TOLERANCE TO THERMAL NEUTRONS 



449 







Fig. 3. Whole brain section of case 8180 r showing a neoplasm producing enlarge- 
ment of the occipital lobe and demyelinization of the adjacent area. The opposite 
occipital lobe and the whole cerebellimi are intact. 



the tip of the frontal lobe and shows part of the scar viewed in Fig. 5. In 
Fig. 7 the photomicrograph shows a section from the scar seen in Fig. 6 and 
is from the outer surface of the brain. The connectixe tissue reaction with 
fibrocytes and the glial reaction are both predominant. Of great importance 
is the fact that within the effective depth no residual neoplastic cells are 
obser\ed in this region. 

Figure 8 shows a photomicrograph from the region about 3 cm deep to the 
gross area of scarring. The neurons are intact, and there is no destructive 
effect of the thermal neutrons in the non-neoplastic area adjacent to the site 
of the previous primary neoplasm. Figure 9 is taken from the meninges at the 
site of the arrow seen in Fig. 6 and within the port of irradiation. The 
meninges are not thickened, although occasionally phaoocytic cells with 
blood pigment arc present. In general, this is a mild reaction, and there is no 
evidence of fibrosis from irradiation. Just below this area, Fig. 10 (a high 



450 



FARR ET AL. 




Fig. 4. Whole brain section of case 8180 r stained to show the Nissl substance. The 
neurons show tigrolysis only in the limits of the neoplasm. 

power magnification with the Nissl stain ) shows intact neurons as further 
evidence of a lack of irradiation efTect. The cortical areas receiving maxi- 
mum exposure received 1,905 rads from capture gamma. This is calculated 
on the basis of the same set of assumptions as the previous values. 



Discussion 

In assessing the role of thermal neutrons as a potentially noxious agent 
when administered to the central nervous system, certain assumptions are 
iinplicit. Among these is the assumption that the only eflFect which need be 
considered in the exposures of the size discussed is that which residts in a 
gamma emission as a result of thermal neutron capture by tissue components. 



HUMAN TOLERANCE TO THERMAL NEUTRONS 



431 




Fig. 5. Photograph of brain at autopsy showing a frontal scar at original operative 
and irradiation site. Case 8-00-83 r. 



452 



FARR ET AL. 




Alio 3 

Fig. 6. Coronal section of gross brain, case 8-00-83 r. at 2 cm from the tip of the 
frontal lobe across the scar partially seen in Fig. 5. 1 here is no tumor growing in the 
region of marginal irradiation. 



While in a few instances the capture reaction will result in an atomic trans- 
formation and a beta particle emission, these are so infrequent that in the 
aggregate no significant contribution to the total exposure is provided by 
them. It then becomes of major importance to determine the formulation by 
which the captiu'c gamma dose is computed. It is here that both the major 
uncertainty and the major difficulty are encoimtered. Since the neutron 
exposure is not uniform, precise description of the attenuation becomes 
paramount. Yet difficulties in measurement and alterations in pattern by 
geometry or port of entry make this factor one of approximation and not 
certainty. In similar fashion, the formulation of a suitable integral which can 
be solved to give the summation of the dose and volume of maximum 
intensity has developed into a problem which can be handled only by a com- 
puter, and we have not obtained a final answer, though work is continuing. 
Therefore, in this discussion we have used the values computed by Robertson 
for the rad dose derived from capture gamma on the assumption of a imi- 
form distribution of thermal neutrons at the maximum observed intensity of 
exposure. It is clear that the dose values given in this paper may be greater 
than the maximum expected value to be obtained by a more precise estima- 
tion, but the volume of maximum intensity in the latter case may equal or 
exceed the approximate value herein reported. Whatever the error and 



HUMAN TOLERANCE TO THERMAL NEUTRONS 



453 













V**? 












.^^ 














S^v* 




Fig. 7. Photomicrograph fX 18) of the scar located in the left frontal lobe, case 
8-00-83 r, showing the thick fibrotic tissue in which there are numerous phagocytes. 
At the left of the brain tissue, lying below the connective scar there is marked gliosis. 



454 



FARR ET AL. 




4-. 



*<Av^ 



?V---_lti >-■ 







Fig. 8. Photomicrograph (X 480) of an area of gliosis located in the white sub- 
stance at a deeper level than Fig. 7. Case 8-00-83 r. 



HUMAN TOLERANCE TO THERMAL NEUTRONS 



455 










''•*r 



» ,'' 












, V 



n 



, 



^fi*^.^^- 









* - 4 



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'^4 . 



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1^ I' 



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?^^- 



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^^ 



















Fig. 9. Photomicrograph C X 150^ of the cortex of case 8-00-83 r taken from the 
zone of maximal irradiation on the frontal lobe below the scar. The cortex appears 
normal. The leptomeninges are not thickened by fibrous tissue. 



456 FARR ET AL. 




Fig. 10. Photomicrograph (X 600) of cortex, case AUG, taken from the same area 
as Fig. 9, showing that the Nissl substance is intact in this zone of maximal irradiation. 



HUMAN TOLERANCE TO THERMAL NEUTRONS 457 

difficulty in calculating the rad equivalent of thermal neutron exposure, we 
have had sufficient experience with measurement of thermal neutrons by 
foil activation to place a high degree of confidence in the total thermal 
neutron exposure indicated. Moreover, the observations, as made, are de- 
pendent on their calculations only for further interpretative elucidation and 
not for primary validity. Preliminary observations on animals and patients, in 
which the maximum intensity reported in these studies has been exceeded, 
are pointed-to only to emphasize that beyond the exposures discussed here 
there still remains a margin of safety. We have been surprised that none of 
the structures, neuronal, supporting, or vascular, have shown any changes 
under the exposures noted. To achieve greater exposures, further change in 
the moderator-reflector system of our reactor must be accomplished, so that 
gammas resulting from materials capture of wayward neutrons may be 
further diminished in a significant manner. 

Since we have observed no evidence of a limit to the tolerance to thermal 
neutrons, we cannot speculate on these data. They are presented as observa- 
tions made repeatedly (in animals) to establish their reproducibility. The 
practical significance of this may be considerable in the consideration of a 
reactor for diagnostic purposes, but until limits are set, this must remain 
largely an area for discussion only. 

It must be emphasized that our observations were not limited to an histo- 
logic examination of exposed tissue. Functional observations were carried out 
on all patients receiving neutron capture therapy. These included specific 
visual acuity and perception tests, audiometric examinations, and complete 
neurologic examinations at frequent intervals. Perhaps the most significant 
was day to day observation of the patient, his general behavior, and his 
reaction to situations frequently new to him. By all these criteria, no dele- 
terious effects were seen. Complex movements were unafTected, and both 
logical and emotional responses showed no changes. Patients were observed 
closely up to I/2 years, which probably is adequate time to permit the de- 
velopment of usual postradiation sequelae. In regard to visual acuity, it 
should be pointed out that frequently the retina itself received considerable 
exposure to thermal neutrons, but no changes in function resulted. 

Summary 

In the histologic studies of serial sections of the brains of 17 patients 
treated by neutron capture therapy for intracranial neoplasms, no changes 
consistent with and attributable to exposure with thermal neutrons were 
found in the nervous tissue. A maximum thermal neutron exposure of 
1.73 X 10^^ thermal neutrons per square cm occurred over a 200 sec interval. 
One additional patient receiving only thermal neutron exposure (approxi- 



458 FARR ET AL. 

mately 4.7 X 10^^ thermal neutrons per square cm to the brain surface) 
showed no histologic changes attributable to the neutron exposure in either 
neoplastic or normal brain tissue. 

The difficulties in transposing the observed neutron exposure intensities 
into rad units are discussed. 

References 

Farr, L. B. 1957a. Les applications mcdicales des reacteurs nucleaires. Bruxelles-med. 
37, 843-858. 

Farr, L. E. 1957b. Present progress in neutron capture therapy. Acta Rad. Interam. 
7, 64-76. 

Farr, L. E. 1959. The Brookhaven medical research reactor. Science 130, 1067-1071. 

Farr, L. E., Sweet, W. H., Robertson, J. S., Foster, C. G., Locksley, H. B., Suther- 
land, D. L., Mendelsohn, M. L., and Stickley, E. E. 1954. Neutron capture therapy 
with boron in the treatment of glioblastoma multiforme. Am. J. Roentgenol. _ 
Radium Therapy Nuclear Med. 71, 279-293. 

Farr, L. E., Robertson, J. S., Stickley, E. E., Bagnall, H. W., Easterday, O. D., and 
Kahle, W. 1959. Recent advances in neutron capture therapy. Proc. 2nd Intern. 
U.N. Conf. on Peaceful Uses of Atomic Energy, Geneva, 1958 .., . See also 
1958, Isotopes in Med. 26, 451-456; 1959, "Progress in Nuclear Energy," Ser. VII, 
Medical Sciences, Vol. 2, pp. 128-138. Pergamon Press, New York. 

Farr, L. E., Calvo, W., Kahle, W., Hochdorph, O., Yamamoto, Y. L., Yakolev, P., 
Lippincott, S. W., and Haymaker, W. 1960. Neutron capture therapy in intra- 
cranial gliomas and sarcomas: Neuropathological study of 16 cases. To be published. 

Godwin, J. T, Farr, L. E., Sweet, W. H., and Robertson, J. S. 1955. Pathological 
study of eight patients with glioblastoma multiforme treated by neutron capture 
therapy using boron-10. Cancer 8, 601-615. 

Lippincott, S. W., Robertson, J. S., Bond, V. P., Cronkite, E. P., Easterday, O. D., 
and Farr, L. E. 1959. Pathological effects of thermal neutrons and of heavy particles 
from the B'" (n, a) Li' reaction in pig skin. A.M.A. Arch. Pathol. 68, 639-650. 

Lippincott, S. W., Yamamoto, Y. L., and Farr, L. E. 1960. Radiation effect of 
neutron capture therapy on a malignant vascular neoplasm of the cerebellum. 
A.M.A Arch. Pathol. 69, 44-54. 



GENERAL DISCUSSION 

Jerzy Rose (Johns Hopkins University, School of Medicine): Many papers 
were concerned with the dose of heavy ionizmg radiation, the consideration of 
species differences, individual differences, differences in different portions of the 
brain, differences in time of appHcation and examination of the tissue, and more 
sensitive methods to detect radiation damage before pathologic changes are 
apparent. Perhaps we need to define a little better what we mean by "minimal 
radiation dose," "lethal dose," and other terms. 

F. A. Mettler (New York, New York): Dr. Malis showed a section in which 
there was irradiation of the Bragg peak and elimination of fibers. Later, on a 
subsequent slide, we saw fibers present in excess. There are three possibilities that 
might explain this. Some of them are rather significant in terms of a possible 
explanation with regard to physiologic experiments in general. The first possibility 
is perhaps trivial, but Dr. Zeman showed a picture in which there was passage of 
the beam and no fibers degenerated. So, the first possibility would be that these 
fibers were not degenerated at all. If one considers the paper by Drs. Janssen, 
Tobias, and Haymaker and several other papers in which there was considerable 
damage in the capillaries in the level of the Bragg peak, the second possibility 
would be that the fibers seen in the second slide of Dr. Malis were really not nerve 
fibers, but were fibers of connective tissue, perhaps fibers of glia. The third pos- 
sibility would be that the fibers seen in the second slide were nerve fibers. If so, 
where did they come from? They could come from two places. There was a paper 
presented earlier which was of remarkable importance to the question of regenera- 
tion of the nervous system. There was transection of the cord with no interference 
with the external meninges, an ideal situation for determination of regeneration in 
the cord. So, these fibers might be regenerative. If this were an area of peripheral 
degeneration or denervation, it would be possible to conceive of another possibility, 
for we know that in peripheral areas of denervation other fibers have a tendency 
to invade. So, if regeneration occurred, it could occur as regeneration from a nerve 
fiber, or it could occur from the invasion of other fibers in the vicinity. Such a 
situation has never been implied in the central nervous system. It it does exist, 
it is of considerable importance in the interpretation of chronic neurologic prep- 
arations. I would like to ask the chairman his opinion. 

Jerzy Rose: We did not present the material completely, because giving evi- 
dence required many slides and there simply was not enough time, so we presented 
only a few slides which illustrated that damage was done. There is no question 
that these are nerve fibers. I think we have rather large material on this aspect. 
If we skip the unessential modification, we have done a sufficient number of a variety 
of these things, and as long as we are going to believe that a silver stain or any 
other accepted classic neurologic stain means a nerve fiber, it is a nerve fiber. Not 
only that, but it is easy to demonstrate in the older lesion that apical dendrites 

459 



460 GENERAL DISCUSSION 

are certain because they are in connection with the cell; of course, it is not always 
easy to be sure it is not an accident. I would say there is no question at all that 
there are nerve fibers and dendrites within the lesion. What are they due to? This 
is an open question. Some people may say that these are preserved fibers. No 
doubt, with a light lesion some fibers may be preserved, but there are many more 
fibers which appear. So, even if you wish to assume that there are no fibers at all 
regenerating, we still have to account for an apparent large increase of the fibers. 
The argument does not rest on the fact that the fibers first degenerate, even though 
there is evidence that they do. There is positive evidence of sprouting fibers, we 
believe. Whether this is regeneration or, as we think, a perfectly normal growth 
which only becomes apparent with light irradiation remains to be determined. 

Law^rence Kruger (University of California, Los Angeles, California): Having 
switched teams, oceans, and cyclotrons, it is a particular pleasure for me 
to be able to say that with lesions of the same size as we had with the Brookhaven 
series, in another species, the rat, and in another laboratory — Dr. Tobias' laboratory 
at Berkeley — Dr. Clemente and I have had excellent correspondence of dosage. 
It occurred to me, however, in listening to Dr. Haymaker, that there was a slight 
discrepance — perhaps an unimportant one. The lowest dosage in the series of Dr. 
Haymaker and his co-workers appears lower than the first animals we irradiated 
in Berkeley which were treated with smaller dosage. We had one animal with 
4,000 rad at 28 days without a lesion, and yet these animals were irradiated on 
the same run. It would seem reasonable to suggest that the size of the lesion could 
explain the discrepancy, since this is the only likely difference in the conditions 
of both sets of experiments. Size is probably important, because Dr. Tobias did 
show an excellent slide of dosage times volume as being an important variable. 
This is also important in discussing the vascular problem in relation to a neuronal 
lesion. Dr. Sourander stated that, with a slit of 1.5 mm and 10 mm, the dosage for 
neuron damage was still 20,000 rad although the appearance was different. I 
wonder whether he would care to comment upon the difference in a general way 
to explain the nature of the dosage volume relationship. Some of the discrepancies 
that might have been apparent at first must be solved in this way. As for species 
difference, it is remarkable that we have a good correspondence now for line 
lesions of the same size for rat, rabbit, and cat. This is now true for independent 
cyclotrons, too. This good agreement in different species does not appear to exist 
for vascular damage, suggesting that neuronal and capillary damage might be 
independent variables. How reasonable is it to assume that the destruction of 
neurons is concerned with the destruction of capillary circulation? I should like 
to join the Drs. Bailey in stating that it does not seem reasonable to say that all 
neuron destruction is the result of capillary damage. To me, the most convincing 
argument is probably the appearance of a line lesion. The sharpness of the lesion 
would in itself discourage the interpretation that this is vascular. However, this 
is certainly not a proof. The collateral circulation might be extensive, and here 
is where the dose-volume problem might solve the question to some extent. A 
recent observation that Dr. Clemente and I have made is that the gross observation 
of capillaries would appear to indicate that the time relationship of the vascular 
change and the neuronal changes in an India ink injected preparation are vastly 



GENERAL DISCUSSION 461 

different. One can see a marked surface change within a time as long as 2 months. 
This at least seems likely at present for a surface dose of 9,000 rad, which is not 
a dose for total necrosis of neurons, and can produce a line lesion in depth. The 
presence of gross capillary changes in a region where neural change is not apparent 
also suggests a possible dissociation of vascular and direct neural damage. The 
gross discrepance between the time relations, the distribution in space in vascular 
changes and the neuronal changes, the gross discrepance between species differences 
and susceptibility of vessels, and the lack thereof for neurons would seem to me a 
convincing argument that neurons are susceptible to ionizing radiation without 
necessarily involving capillary damage. 

Horace W. Magoun (University of California, Los Angeles, California): I want 
to ask another question related to this same program of study, which in addition 
to its importance for central neural regeneration would seem to be a great po- 
tential for contributing to the neurophysiology of the lamina of the cerebral cortex. 
Here is the first time it has been possible to interfere morphologically with 
individual cortical laminae. One of the most interesting developments that has 
come in this area recently is the concept that one can identify in neurons two types 
of excitation: the classic conducting all-or-none mechanism in the axon and a 
graded response mechanism with a longer time course and excitable only to 
chemical transmitters at the synapses on dendrites. I judge that these laminar 
sections sever the apical dendrites, at least, of deep-lying cortical pyramidal cells; 
and for a time before they regenerate (if that is what they do), the distal apical 
dendrite must be destroyed. A whole category of evoked potentials recorded from 
the surface of the cortex have been attributed to this graded response mechanism 
of the dendrites, and they are called, perhaps loosely, "dendritic potentials." Among 
these are the surface-negative local cortical response, the recruiting response from 
exciting the nonspecific cortical projection, the surface chemical features of the 
augmenting response to the repetitive excitation of specifically projecting terminal 
cortical connections, and features of the transcollosum potential. Because each of 
the co-authors of this remarkable contribution is a sophisticated neurophysiologist 
as well as morphologist, I wonder if one of them would tell us what is being done 
in the study of electrophysiology of this situation, which it seems to me is far more 
interesting than simply the morphologic observation, and in particular what is 
happening to what is being called today "cortical dendrite potentials." 

Leonard I. Malis (Mount Sinai Hospital, New York, New York): Again 
as in the growth of ner\'e fibers, the story is long, and we cannot go into detailed 
presentation. Briefly, we have prepared 75 or so cats with unilateral right striate 
cortex laminar lesions at various layers and at various times after the irradiation 
have studied them with surface maps and with microelectrode punctures for the 
evoked responses in striate units above and below the laminar lesions to light flashes 
in optic stimuli. Unfortunately, the study required a great deal of correlation with 
the various times of the units, and with silver stains to show us the stages of fiber 
regrowth. We are in the process of analysis now, and some of the changes have been 
somewhat surprising. One of the most difficult to do was to get the units to stop 
firing on top of the lesion. They have a remarkable tendency to continue this. The 
surface maps showed almost consistently, whenever the laminary lesion was fairly 



462 GENERAL DISCUSSION 

decent and not totally necrotic, a marked increase in the evoked response just 
anterior to the lesion from a band of several millimeters, as compared to the 
normal side. Relatively thin lesions may have an increase of activity to the evoked 
response technique even within the lesion. The heavier lesions may have (in the 
earlier phases at least, before the nerve fibers are all back) a considerable decrease 
within the lamina, but still will have a hyperactive zone in front. 

Lal Harbans (University of Chicago): It was pointed out in various papers 
that radiation produces changes in the blood-brain barrier, and an increase in the 
mobility of the blood vessels can be produced. In studies on various effects on 
nerve and other tissues, it is possible that the effects were altered by an animal 
kept for observation which is eating toxic substances which reach the nerve cells 
and other tissue in greater concentration because of the barrier changes. Thus, 
we are exposing the tissue to greater toxicity. In one of the papers on the blood- 
brain barrier, a dye was used, and it was only possible to show changes with a dose 
of about 5,000 r in monkeys and only in those areas which already have soft 
barriers, such as those close to the cerebrospinal fluid and around the hypophysis. 
The sodium level was changed in doses as small as 15 r. When dyes are used as 
indicators, they have a strong tendency to bind themselves with plasma proteins 
which are large molecules. You should be able to make really big holes in the 
blood vessels so that these proteins, along with the dyes, should be able to per- 
meate and show on the slides. Using sodium and other smaller organic molecules, 
as we use in labeled form, we keep in mind their various degrees of binding with 
plasma proteins. If one can decrease the binding and let the molecule be small 
enough to go back and forth by itself, one may be able to pick up smaller changes, 
which will not be possible if you have not pushed up the plasma proteins through 
the blood vessel to indicate the changes. 

Leo E. Lipetz (Ohio State University): In regard to the question Dr. Kruger 
raised earlier about how a vascular change could cause such sharp destruction 
of the neurons, I would like to do a little speculating in terms of the retina. 
In the retina, every neuron is surrounded by glial cells, and apparently it 
has to get all its nutrition via the glial cells. The glial cells are not large, so by 
injuring them you could produce a localized destruction or interference in the 
nutrition of a localized band of neurons. This might be a means of producing local 
neuronal damage. I wonder whether this could apply to the rest of the central 
nervous system. 

Orville Bailey (University of Illinois): I would not think that these lesions 
could be well explained on neuroglial injury alone. Dr. Rose gave me the oppor- 
tunity of spending some time on his preparations. The destruction of neuron 
elements in the band is complete, and it is incredibly sharp. Along the edge of the 
band one occasionally can see a cell body minus its apical dendrite in the more 
acute phase. In the reparative phase, similar cells are so placed that it seems 
necessary to conclude that the dendrite has grown back. From the standpoint of 
general neuropathology, the evidence of true regeneration of neurons is of two 
types. There are too many nerve fibers in the area. As a general pathologic phe- 
nomenon, repair tissue ought not to be exactly in the expected normal proportions. 
It is quite often too much, a criterion fulfilled admirably here. Second, the orienta- 



GENERAL DISCUSSION 463 

tion of fibers ought not to be quite according to the normal pattern. And here the 
striking thing is the orientation at an angle — often at a right angle — to the expected 
one. The histologic technique is such that it leaves no question that these are truly 
nerve fibers. In fact, the technique is so good as to give one every confidence in 
the care with which the whole investigation has been carried out. 

Webb Haymaker (Armed Forces Institute of Pathology): One of the questions 
was directed to me with regard to the fluorescent technique data by Dr. Calvo. It 
was suggested that there perhaps were some holes knocked in some of these vessels, 
and the extrusion of the labeled albumin came through these holes. To that I 
would say this: This was a 6,000 r surface dose, and the first changes were seen 
in two days. At that time, under the light microscope, we could find no mor- 
phologic alterations in the blood vessels. Secondly, there were no hemorrhages. On 
the basis of this and other data, I believe that Dr. Calvo would feel that this is 
really a matter of permeability to a rather high molecule. I do not think there is 
any way of comparing the experiments of Dr. Van Dyke, using soluble fluorescein, 
with the technique which we use. His lesions were made with a 184 in. cyclotron 
with far greater energy. I would suspect that if he had used 6,000 rad and 
duplicated our experiment the soluble fluorescein would come through much more 
rapidly than the protein molecules, the data on which we presented. 

Cornelius A. Tobias (University of California, Berkeley, California): First, I 
wish to comment on the remarks Dr. Kruger made with respect to the dose-eflfect 
relationship. You could see from the curve I presented, which had the dose volume, 
that not all the points were exactly on the curve. Actually the relationship is 
probably much more complicated than that. If you keep the volume the same and 
give different doses, you have one relationship in which with lower doses, the 
lesions appear later. Also, as Dr. Sourander has done, you can use the other ap- 
proach, keeping the dose constant and changing the volume. This will give rise 
to another curve. So we have actually more than one parameter to deal with. 
Moreover, I believe now that different parts of the brain are somewhat different. 
For example, in the cerebellum you get a slightly different dose-effect relationship 
from that in the cerebrum. However, when you try to plot the relationship to the 
time, the time slope of the curves obtained is about the same; whereas, the abso- 
lute position of the curve in the dose-effect relationship is somewhat displaced. I 
think we have heard possibly enough evidence today to realize that there must be 
at least two different relationships. The relationship where the dose and time both 
enter makes one strongly suspect the intereferency or the importance of circulation, 
and of the capillarity and profusion by blood, of vascular accident, like hemorrhage 
that may occur on a statistical basis. On the other hand, some of the data pre- 
sented with respect to neurons have a more or less direct inner dose-effect rela- 
tionship, as in the nuclei that Dr. Janssen counted. It may be due to a direct 
effect, perhaps possibly even a direct effect of particles which hit the nuclei of the 
cells. Dr. Clemente of U.C.L.A., Dr. Richards of our laboratory, and Dr. Gaffey 
have undertaken ambitious programs, in which electrical activity will be studied 
as the function of the location of the irradiation and the dose. The technique used 
is imbedding electrodes in various locations, then placing the lesion with the high 
energy beam at the various locations. For example, at the top of one of the sets 
of electrodes. Then it probably is possible to stimulate and study electrical activity 



464 GENERAL DISCUSSION 

in various parts of the brain with regard to accessibility. The Swedish investigators, 
particularly Dr. Anderson, have for many years been studying the hypothalamus 
with electrodes which reach in the same direction. And the Russian investigators, 
in total brain irradiation studies, have for some time been realizing the inacces- 
sibility of the hypothalamus which follows irradiation. 

Jerzy Rose: It seems to me that as far as protons, alpha particles, and neutrons 
are concerned there seems to be agreement among all hands as to the minimal 
dose, properly defined, with a proper relationship of latent periods and without 
fluorescein. Minimum lethal dose seems to be about 50,000 rad. The total destruc- 
tive dose is around 75,000, an unsatisfactory figure in detail, but of the proper order. 
Perhaps our Swedish guests would comment on whether they agree with this con- 
clusion and Professor Grashchenkov would comment on what the investigators in 
the Soviet Union found. 

N. I. Grashchenkov (U. S. S. R.): We have some figures which indicate that 
part of the brain connection with the hypothalamic region was damaged by a 
small dose of irradiation. At the same time we have some investigations — mor- 
phologic as well as postphysiologic — which indicates the role that the brain played. 
In our country, Professor Stamm and his large school with many collaborators 
have dealt with this problem. In this last period, his studies indicated that this 
formulation played an important part in the mechanism of hyalinization. 

Patrick Sourander (University of Gothenburg, Goteborg, Sweden): When the 
spinal cord was irradiated with the 1.5 mm beam, there was little vascular damage 
seen. Of course, there was irradiation of red blood corpuscles in the capillaries, 
but no damage of the bigger cortexes. If a broader beam was of 10 mm, after a 
few days considerable damage of blood vessels was seen with massive hemorrhages. 
One explanation would be that with the 1.5 mm beam only a relatively small 
volume of capillaries are destroyed, and there might be compensatory mechanisms 
in the circulation. When a bigger volume is destroyed, this compensatory mech- 
anism fails. I do not know if this is the right explanation, and I would like to 
ask the specialists what they think about such an explanation. The other phenom- 
enon observed, both in the thin beam and the broad beam, was that some fibers 
were always affected. One possible explanation would be that the situation as to 
the blood supply might be better for the periphery or the spinal cord than the 
central parts. Concerning changes in the nerve cells, I would like to mention a 
general point of view. I doubt the reliability of the methods used for studying the 
nuclear proteins and nucleic acids in nerve cells after irradiation, whether used in 
fluorescein stains or aniline stains at a certain pH. Certainly, there are more 
reliable methods, such as the ultraviolet absorptions at 2,600 Angstrom, both 
before and after digesting with ribonuclease or desoxyribonuclease. There are 
recently published methods from Goteborg concerning nucleic acids seen in the 
glial cells. They were studied by means of electrophoresis on a micro scale with 
single cells. It is by no means clear that there should be initially a depletion or 
reduction of nucleic acids in degenerating neurons. As was recently shown by 
Ingstrom, after the axon is cut, there is an increase of nucleic acids and later a 
decrease of nucleic acids. I think these newer methods should be used when 
studying the effects of irradiation on nerve cells. 

Herbert Locksley (Iowa City, Iowa): I have been appreciative of the progress 



GENERAL DISCUSSION 465 

reported to us by Dr. Haymaker, Dr. Tobias, Dr. Van Dyke, and Dr. Clemente on 
the difficult problem of trying to quantitate irradiation changes in the blood-brain 
barrier. To emphasize the point made by one of the discussants, most of the dyes 
that are used are bound by serum protein, and therefore changing permeability to 
them represents a gross breakdown in the barrier. One question for Dr. Clemente: 
In the classic descriptions of the use of trypan blue, it has been observed that 
normally some areas of the brain are permeable to trypan blue, namely, the 
superoptic and infundibular regions, the area postramus and the locus cinereus in 
the fourth ventricle. It seemed to me from your shdes that these areas were 
strongly represented in those shown after low dosage radiation. I had the privilege 
of participating with Dr. Lee Farr and Dr. William Sweet in the second series of 
neutron capture radiation at Brookhaven, a series of 10 radiations, to study intra- 
cranial neutron flux during treatment and to make some physiologic studies of 
possible changes in the blood-brain barrier. This is of crucial importance in that 
type of treatment because its success is contingent on a ratio of concentration of 
boron or other capturing agent within the tumor cells and in normal tissue. With 
boron, this has been established to be about 3 or 5 to 1. The high ratio is main- 
tained for a short time, perhaps half an hour, which limits the duration of 
radiation. The question that arose was this: Does the patient after one treatment 
have sufficient changes in the blood-brain barrier to reduce the therapeutic ratio, 
that is, to reduce the concentration between tumor and brain, which might com- 
promise subsequent treatments in the same patient? We considered the possibility 
of using dyes, but felt this would represent too gross a test. Extending some tech- 
niques for studying the dynamics of cerebrospinal fluid formation and absorption 
developed by Dr. William Sweet and myself, simultaneous isotopic tracer studies 
were made in patients with tumors before and after neutron capture radiation 
using isotopes K-24, sodium-24, heav^ water, P-32 and chloride-38. In patients 
who had received possibly 1,000 to 3,000 rep of neutron capture therapy, there 
was no consistent detectable change in the blood cerebrospinal fluid barrier to 
these isotopic tracers. 

Ray S. Snider (Northwestern University): I found this afternoon's group of 
papers stimulating. I don't think I have ever sat through a half-day session among 
the radiologists and found it more rewarding from the standpoint of getting new 
ideas for future experiments. That is a bold statement, even for a chairman to 
make. I would like to point out that we are dealing here with an exquisite 
microradiologic tool that can destroy not only single cells, but even parts of cells. 
I would like to ask what happens when this radiation goes in a cell and tickles it 
gently without destroying it. I ask that because of the microchemical methods 
now at our disposal, especially those of Lowry and associates, where they do 
chemical reactions on single cells and parts of cells. I also ask it because of the 
newer electron microscopic methods that we have, especially in relation to the 
study of the structure of protein membranes. I ask it because of Dr. Magoun's 
question on neurophysiologic activity, where we are measuring activities of single 
cells, parts of cells, and the cellular environment. I ask it from the standpoint of 
regrowth of the cell and emphasize again a point made by Dr. Mettler. I would 
like to know what is happening to the behavior of this cell not only electrically, 



466 GENERAL DISCUSSION 

but also from the standpoint of the chemical environment. I could not help but 
agree with Dr. Lipetz when he raised the question — what is happening to the 
neuroglia here? We have long wondered whether or not they may help to 
conduct the nervous impulse. This is an excellent method, it seems to me, for 
answering this old question. It is also a good method to us for the study of 
environmental vascular changes. 



PART IV 



Functional Changes in the 

Nervous System Resulting 

from Radiation Exposure 



Review of Neurophysiologic and Psychologic 
Research on Irradiation Injury in the U.S.S.R.* 



Walter R. Stahl 

Oregon State College, Corvallis, Oregon, and 
University of Oregon Medical School, Portland, Oregon 

One often hears the statement, "Using conditioned reflex techniques, 
Soviet scientists have found the central nervous system to be very sensitive to 
ionizing radiation." The goal of this analysis is to demonstrate that such a 
statement is an oversimplification ; it requires considerable elaboration before 
a clear interpretation of the implied claim is possible. 

Most of the illustrations to be cited are drawn from the Soviet radiobio- 
logic literature and cover materials through August, 1960. An extensive 
bibliography has already been presented in two recent review papers (Stahl, 
1959, 1960). However, several sources for the scientist who does not read 
Russian have been cited, such as reports for the United Nations Scientific 
Committee on the Effects of Atomic Radiations (Brazier, 1959; Bykov, 
1957; Gubin and Moskalev, 1960; Livanov and Kondrat'yev, 1960; Pavlov, 
1959). 

This review attempts to distinguish several fundamentally difTerent nerv- 
ous system reactions which may take place after irradiation. These include 
(1) minor damage to neurons, not inconsistent with the phenomenon of 
"radiation aging," (2) specific or selective damage to the central nervous 
system (CNS) causing loss of particular ner\e functions, (3) perception of 
radiation by the CNS without any pathologic consequences, (4) abnormal 
CNS functioning, resulting from radiation, which causes or contributes to 
radiation sickness, (5) somatic mechanisms which alter the response in a 
test of CNS activity in a misleading manner, and (6) certain general reac- 
tions, such as alterations in levels of circulating biochemical mediators 
which result in altered nerve functioning. 

In addition, it is always necessary to bear in mind the age, species, and 
sex of experimental animals, the size of the radiation dose, whether radia- 
tion sickness actually developed, how long after exposure the CNS test was 
done, possible disturbances in the general state of the animals, such as 
change in appetite and normal motor activity, and validity of the result, 
statistically. 

* This paper was presented at a dinner meeting between the third and fourth 
sessions. 

469 



470 WALTER R. STAHL 

Radiation Damage which is Histologically Apparent 

As is well known, beyond a certain dose nerve tissue shows major damage 
with cytolysis, fiber degeneration and eventually scarring. In the Soviet 
literature, such pathology has been reported to occur after 1,500 r in some 
cases, though doses of 5,000 r are often mentioned also as the threshold for 
really major damage (Livshits, 1956; I. A. Brodskaya and Merkulova, 1956). 
A demyelinization syndrome following several thousand r was cited recently 
by Bibikova (1959). 

However, many Soviets have found injury at levels far below these. For 
instance, Lebedinsky (1959a, b; Lebedinsky and Moskalev, 1959) describes 
rather selective injury to the cortex, hypothalamus, certain autonomic struc- 
tures, and elsewhere following near-lethal mammalian doses. Selective 
damage to afTerent neurons of the spinal cord has been reported by several 
workers, such as Shabadash et al. (1959) who used a special RNA isoelectric- 
point staining technique. Others have found autonomic, and especially sym- 
pathetic peripheral structures, to be most sensitive (Smirnov, 1960). Spinal 
synapses were found partially damaged following exposures to 450-600 r 
(Lev, 1957). 

In these studies, injury was fairly general in the structures cited. Scattered, 
random injury with quite low doses has been reported by Soviet workers. For 
instance, Aleksandrovskaya (1958) found an increase in numbers of patho- 
logic cells after only 50-150 r, with 150 r also damaging a few glial cells. 
Spotty damage to peripheral nerve has been cited after a few hundred r 
by Garvey (1960), Anisimova- Aleksandrovskaya (1959), and others. 

Many Russian papers have dealt with postirradiation injury to nerve 
structures within specific organs. For instance, Zarat'yants (1960) states that 
only 60-90 r of chronic exposure induces alterations in the nervous structures 
of the gastrointestinal tract; this pathology is said to be the first seen under 
the cited exposure conditions. The extent of injury was small. Levinson et al., 
(1957) could find only slight injury in nerve structures of the gut following 
3,000 r. In skin structures of rats exposed to 700 r (Garvey, 1960), changes 
were slight during the latent period of the radiation syndrome, marked at 
about 5-20 days, and still evident as late as 60 days after exposure. Damage 
to cutaneous nerve structures only 30 minutes after irradiation is noted by 
Oleynikova (1959). Gromada and Polachek (1959) found significant injury 
to receptor endings in heart muscle, fascia, and elsewhere after many hun- 
dreds of r. Major injury of nerve endings in the spleen and lymphatic organs 
following near-lethal doses have been reported by various Soviet workers 
such as Alekseyeva (1958). 

Scattered, low level pathology is not easy to demonstrate. It is evident that 
the researcher must use extensive controls and analyze his results statistically. 



REVIEW OF RESEARCH IN U.S.S.R. 471 

Allowance must be made for possible tissue autolysis after spontaneous deaths 
and for intercurrent intoxication, focal or general infection, hemorrhagic 
diathesis, fever, and other well known concomitants of acute radiation sick- 
ness. Thus, a clear and explicit statement must be made about when the 
injuries were seen in respect to occurrence of an acute radiation syndrome. 
Changes in the nutritional state of the animal and use of any drugs must be 
considered. Development of low level viremia is entirely possible after sub- 
stantial radiation exposures and would be expected to cause CNS reactions. 
Interpretation of nerve injury in specific organs, such as the spleen, is com- 
plicated by the extensive cellular changes known to take place in this struc- 
ture after radiation. The extensive cellular restructuring seen after exposure 
may cause reactions in nerve endings. In general, most of these papers find 
much more significant damage during the acute radiation syndrome, with 
only reactive or irritative changes in the latent period. Effects on receptors 
are of great interest from the standpoint of possible abnormal afferent flow to 
the CNS during developing radiation sickness. 

Soviet and Western workers agree that the very young CNS is much more 
radiosensitive than the developed CNS. By way of illustration, Olenov and 
Pushnitsyna (1952) find extensive neuronal injury in young animals after 
only 40-120 r. Kosmarskaya and Barashnev (1958) describe reactions in 
neonatal rats exposed to 250-500 r. Others have described radiation-induced 
congenital anomalies in the CNS. 

In regard to histologically apparent findings, one can say that while scat- 
tered, random injury to some nerve structures would not be too surprising 
following several hundred r, there is a heavy burden on the experimenter to 
prove whether this is direct or due to somatic radiation injury. He must also 
control and eliminate all possible secondary mechanisms known to injure 
neurons, such as fever, viremia, hemorrhage, and general cachexia. In the 
absence of full details on control measures, reports on low level neuronal 
injury are hard to interpret. 

Functional Damage to Nerve Structures which is Not Apparent 
Histologically 

Important changes may take place in synapses, neuronal membranes, and 
elsewhere, but not cause any notable alteration in histologic preparations. 
Because of well known difficulties in the electron micoscope study of the 
nervous system, the discovery of random minimal injury is not going to be 
easy. One can cite a great many possible mechanisms of nonmorphological 
radiation damage. 

Several papers have dealt with direct radiation effects on simple nerve- 
muscle preparations. For example, Pshennikova (1958) reported both local 



472 WALTER R. STAHL 

damage and spreading axonal effects from a small segment of frog sciatic 
nerve exposed to 10 kr and more. Lebedinsky (1959b) assessed synaptic trans- 
mission in the irradiated animal under physiologic conditions by testing 
blinking of the eye following cervical sympathetic stimulation; alterations 
occurred after doses of a few hundred r. 

In another synaptic study, using a leg withdrawal reflex in the rabbit, 
Kudritsky (1957) found that 10 r will alter some of the timing parameters, 
especially the variability of the latent period of response. Similar types of 
alterations have been reported following 500-1,000 r (Gvozdikova, 1957). 
Kudritsky has also suggested that there may be adaptation of the organism 
to radiation and found evidence that pretreatment of a rabbit with several 
hundred r eliminated its response (noted previously) to a subsequent 10 r 
test. 

Many indirect factors can enter into an altered CNS response following 
irradiation such as changes in basic metabolic processes in nerve cells in- 
volving DNA metabolism (Levinson et al., 1957) or cerebral amino acid 
metabolism (Minayev and Skvortsova, 1957). No final conclusions seem 
possible on this point; nucleic acids are known to be fairly radiosensitive 
in some biologic systems, but maintenance of the membrance potential is 
expected to be quite stable to slight biochemical damage. 

Many Soviet studies have dealt with various vascular reactions to radi- 
ation exposure. Lyubimova-Gerasimova (1960) found significant alterations 
in tone of cerebral vessels following 1,000 r in the rabbit. Several studies 
discuss blood-brain barrier permeability changes and alterations in capillary 
properties; these may well play a role in some of the reactions seen after 
exposure. 

The possible importance of circulating toxic or physiologic substances in 
radiation reactions has been stressed in many works. Working under Lebe- 
dinsky, Maslova (1958) and others have produced evidence indicating fluc- 
tuations in circulating sympathin levels following near-lethal exposures in 
cats and rabbits. There seems little doubt that under some conditions sym- 
pathins are increased and that they fluctuate considerably. These substances 
may well exert a role on almost any aspect of CNS activity. The possible 
presence of sympathins should be given serious thought in experimental 
results without a ready interpretation, such as atypical subcortical EEG 
reactions. Mozzhukin and Pevsner (1959), working under Zedgenidze, 
showed important changes in pressor reflexes following radiation and sug- 
gested that altered adrenal medullary activity was responsible. Hypotonia 
or other vascular reactions may alter reactions in CNS tests. 

Direct radiation injury to an effector organ needs serious thought with 
CNS studies involving secretory organs. For instance, Lomonos (1957) and 
Ye. A. Brodskaya (1958) showed that there are changes in the uncondi- 



REVIEW OF RESEARCH IN U.S.S.R. 473 

tioned responses of the salivary glands and gut secretory organs following 
radiation, a fact which would be anticipated from the known postexposure 
histologic reactions of these organs. Thus any conditioned reflex study using 
a secretory response is difficult to interpret. 

One would hope to eliminate such difficulties by using motor studies in 
experimental animals. These are usually based on feeding reflexes, however, 
and it is well known that irradiated animals show alterations in appetite 
and spontaneous motor activity. They may develop an aversion to certain 
foods for no known reason and may specifically avoid foods eaten shortly 
before irradiation (Kimeldorf, 1961). Following anorexia, irradiated ani- 
mals may develop an appetite which is stronger than normal, and this may 
lead to important alterations in some psychologic testing situations. 

Conditional Reflex Studies 

The term "conditional" rather than "conditioned" is used in this report 
because it represents a better translation of the corresponding Russian word ; 
it has been advocated by a good many U.S. conditional reflex (CR) re- 
searchers because it stresses the temporary or "noninnate" nature of CRs 
(Brazier, 1959). 

Some assumptions concerning CRs which are generally taken for granted 
by Soviet physiologists may be little known to Western radiobiologists. The 
serious student of CR testing is urged to read Pavlov (1959) — there is now 
available an excellent translation by Anrep — and the recent review of CR 
studies by Grashchenkov (this volume) . At present Soviet physiologists often 
use motor-feeding reflexes and include an instrumental response in their anal- 
ysis. Thus, the typical modern CR test technique may be quite difTerent from 
that using a dog in a harness and approximates a total-observation situation 
of activity in an animal, as presented with a sequence of stimuli. In some 
cases work has been done with dogs running free in a room, but more 
commonly rodents in a small sound-proof chamber are used. Under these 
conditions one can get information about the spontaneous motor activity of 
the animal. However, CR studies do not lend themselves to tests of spon- 
taneous preference, a technique which Western radiation researchers have 
shown to be sensitive in postradiation experimentation. 

The magnitude of the response to the presented stimulus is of cardinal 
importance. Pavlov demonstrated the important basic fact that a strong 
stimulus produces a quantitatively larger response, as exemplified in measure- 
ment of drops of saliva. The same holds true for motor reactions, as shown 
in the force exerted by the animal. Typically, the animal is presented with 
a fixed sequence of stimuli of varying strength (a "stereotype" of stimuli). 
For example, one can use a sound of a certain frequency as the "strong" 



474 WALTER R. STAHL 

stimulus, a weak light as the "weak" stimulus, and a sound of the same 
intensity, but different frequency, as the "differentiating" or inhibitory 
stimulus. 

In a normal animal with a well developed CR pattern, there is a clear 
parallelism between the strength of stimula and response, and differentiation 
is distinct and complete. Possible alterations in CR stereotypes include 
equalization of reactions to varying stimuli, or a strong reaction to a 
weak stimulus and none at all to a strong one, failure of differentiation, 
weakening of all responses, residual inhibition following the differentiation 
stimulus, or increased errors or variability of response. All of these changes 
have been stated to occur after radiation, in Soviet studies. 

Pavlov demonstrated that animals differ in their reactions to a stereotype. 
He classified several higher nervous types (CNS types), namely, strong- 
balanced, strong-unbalanced, weak, and poorly balanced types, the last 
with a predominance of excitation or inhibition. In all cases, one is referring 
to the occurrence of excitation and inhibition, as judged by strength, balance, 
and mobility. Objective methods for classifying animals are well known in 
the Soviet Union and involve tests of reactions to strong stimuli, reactions 
to inhibitory stimuli, responses following caffein, etc. Typing is not always 
easy or clear-cut, and many animals may not fall into a distinct type, but 
nonetheless this classification is helpful for assessing reactions. In higher 
animals the complexities are still greater, but human typing has been 
attempted. As a simple illustration, persons react differently to a strong, 
frightening stimulus — some tend to respond with activity (excitation) and 
others with inactivity (inhibition) ; moreover, the time needed for restoration 
of normality varies (being a function of CNS mobility and balance). The 
occurrence of CNS types adds another degree of freedom to CD studies 
and complicates their interpretation further. 

Pavlov did not attribute any morphologic or biochemical basis to excita- 
tion and inhibition but felt the phenomena were purely functional. Basically, 
one starts with the view that salivation is preceded by excitation in the 
"salivatory center," and that there are antecedent foci of excitation before 
the latter is stimulated. If salivation fails to occur when expected, then 
inhibition occurred somewhere in the cerebrum. The quantitative nature of 
CR experiments places a strong emphasis on the relative balance of excita- 
tion and inhibition. Possibly the terms excitation and inhibition have been 
identified too fully with the identical words used in Western psychology, 
although in the original Pavlovian context they were different and perhaps 
simpler. 

"Internal inhibition" often enters into the discussion in studies of radi- 
ation effects on CRs. Pavlov originally distinguished between types of inhi- 
bition, such as that exemplified by failure of a response due to frequent 



REVIEW OF RESEARCH IN U.S.S.R. 475 

monotonous repetition of the stimulus and that due to a sudden distracting 
stimulus, as a door slamming. Later, however, he expressed doubts about a 
precise classification of inhibitory efTects. Internal inhibition is typically in- 
nate inhibition which develops in the nonnal course of training, as with im- 
proving differentiation. It is said to be often the first function to show 
damage following any noxious influence, including radiation exposure. In 
my view, internal inhibition can be compared with a multiplicity of cyber- 
netic functions in the CNS, functions which refine the accuracy of CNS re- 
actions and control them. 

Finally, statistical analysis of CR studies is rather complex. Theoretically, 
it typically involves the interpretation of a 8-dimensional vector (the stere- 
otype) in all its possible alterations, a problem which does not lend itself 
to simple treatment. One may try to isolate certain elements, such as the 
strength of the strong positi\e response or the degree of difTerentiation, 
and test them statistically, but this would not necessarily prove or disprove 
that some subtle change has occurred in the entire stereotype. Under these 
conditions, it is easier to understand that the opinion of the experimenter, 
who has typically done hundreds of studies on each of a small group of 
animals, is accepted at face value by most Soviet physiology laboratories. 
However, more objective methods of evaluation will clearly have to be 
developed, and their design represents a considerable challenge for proba- 
bility theory. 

In studies of radiation effects on CRs, Soviet physiologists have found 
differing reactions, and no final opinion concerning the radiosensitivity of 
the CR mechanism ought to be reached at this time. 

Some workers have observed subtle changes in CR patterns following 
very low exposure. It was reported to the UNSCEAR that 50 mr had some 
effect on an inhibitory CR, interpreted as being due to a change in internal 
inhibition. Several papers by Piontkovsky (1959) and Khozak (1958) state 
that 0.5-20 r single exposures have a stimulatory effect on conditioned 
responses lasting days or even months. Cherkasov ( 1960) asserts that a single 
dose of 30 r or chronic exposure at the rate of 0.1 r per day may cause 
disappearance of all CR responses in certain experiments. Others state 
repeatedly that CRs show changes after only low radiation exposures. In 
rats having a motor-feeding CR to successsive 50 r doses, with interv-als of 
1-3 weeks for a total of 350 r, Khozak (1958) found marked variability in 
the particular animal following only 50-100 r total dose (Fig. 1). He inter- 
prets this result as primarily a stimulatory and disinhibitory action on higher 
nervous activity. It is unfortunate that only one control graph is shown, 
since the variability of the animal response prior to irradiation is critically 
important. 

Another group of experiments deals with near-lethal exposures in rodents 



476 



WALTER R. STAHL 



200 
180 
160 
140 
120 
100 
80 
60 
40 
20 




Preradiation 



I 






28 




|] 






32 



53 



91 



Fig. 1. Alterations in conditional positive and inhibitory reflexes following exposure 
to 7 doses of 50 r each. The first graph gives control data: irradiation was administered 
as shown by the word written vertically, and the time scale is in days following each 
day of administration. This animal is considered strong and well balanced, and the 
upset of strength relations and frequent increase in the motor-feeding reaction is noted 
particularly. Redrawn from Khozak (1958). 



REVIEW OF RESEARCH IN U.S.S.R. 477 

and dogs and is typified by the work of Livshits (1956), Yarullin (1959), 
and Lomonos (1957, 1959). These experiments may show a slight stimula- 
tory efTect initially, but more commonly they show depression of the CRs 
a few weeks after exposure. The possibility of an indirect somatic effect, 
such as suppression of salivatory activity, must enter into the interpretation 
of these findings. Most studies suggest depression of CRs after several 
hundred r, at some time after exposure. 

However, quite a few Soviet authors, such as Korol'kova (1958) and 
Biryukov (1957), noted slight or insignificant alterations in certain CR 
experiments after 1,300-5,000 r (chronic exposure, usually). It is doubtful 
that these authors would deny any effect on a CR with low doses, but in 
some studies they were able to produce quite normal new CRs even after 
thousands of r. 

Thus, it is apparent that there is no unanimity at present as to whether 
the CR method shows very low level radiation alterations or whether it is 
a highly sensitive indicator of damage. More work will have to be done on 
this matter, and it will have to be fully controlled statistically; the interpre- 
tation must differentiate between direct effects on the CNS and indirect 
actions through somatic organs. 

Other types of tests of general higher nerve functioning after radiation 
include studies on experimental neuroses following exposure. Korol'kova 
(1958), workers at the Sukhumi primate laboratory in the U.S.S.R. (per- 
sonal communication), and others indicate that experimental neuroses 
develop quicker, are more severe, and last longer after irradiation. In a 
general way, some researchers indicate a "weakening" of higher nervous 
activity, particularly in regard to inhibitory control. Livanov (1957, 1959; 
Livanov et al., 1960a, b) has stressed repeatedly that such injury may be 
hard to detect because the CNS compensates for the insult almost immedi- 
ately; he feels CNS radiation damage is nonthreshold and cumulative. 

Substantial portions of the cortex have been removed in experimental 
animals without drastic, sometimes without any discernible, effects on CR 
and behavioral tests. These techniques would hardly be expected to reveal 
damage to a small percentage of neurons in the cortex or elsewhere. One 
can probably speculate that negative feedback, compensatory mechanisms 
are present in all parts of the CNS, as are nonlocalized pattern transfer 
systems. As pointed out by von Neumann, one can make reliable devices out 
of unreliable components by multiplexing circuits, and, therefore, it seems 
that slight damage in the CNS will be exceedingly hard to demonstrate by 
ordinary methods. 

Special test methods may turn out to be of the greatest interest from this 
viewpoint. For example, one may mention the report on audiogenic seizures 
in mice, presented at this conference, and work by Biryukov (1957) showing 



478 WALTER R. STAHL 

that 100 r to the head of fowl in a state of catalepsy would reproduclbly 
arouse it in a condition of agitation. Elsewhere it has been observed that 
sleeping animals may be awakened by modest radiation doses. Humoral 
mechanisms should not be ruled out in these cases, and indirect actions, 
such as variations in blood-brain barrier, should be kept in mind when 
interpreting the results. 

Certain types of CR studies are infrequent in the Soviet literature. There 
seem to have been no tests reported in which radiation was the conditioned 
stimulus (e.g., a signal of impending shock) which might indicate the lowest 
perceptible dose or subliminal effects. Work on conditioning of somatic 
effects is limited. Some older articles indicate the p>ossibility of conditioned 
leucopenia, hyperglycemia, and other reactions, but the results do not seem 
to have been confirmed. The possibility of humoral changes would have to 
be kept in mind with such studies. Radiation has been used as an uncondi- 
tioned stimulus by Kimeldorf (1961), but in this paradigm it served as 
an accessory factor modifying the thirst reaction. 

Much interesting speculation is possible as to the basic mechanisms which 
may be involved in the typical CR and behavioral tests. CR trials typically 
do not involve learning; the animal knows what the signal means, and the 
response to the known stimulus is the matter of critical importance. Even 
in differentiation trials, it is normally not a question of learning, but of 
discriminating between two known alternatives. Nor do CR studies deal 
with frequency of response, which is determined by the experimenter. They 
might be loosely considered to be primarily motivational tests, but with the 
added important factor of residual influences from the previous elements 
of the stereotype. The full analysis of correspondence between various types 
of psychologic tests used in the U.S., Europe, and the U.S.S.R. is a matter 
that deserves more attention. 

Nervous Mechanism in Radiation Sickness 

Livanov (1957, 1959; Livanov et al, 1960a, b) has been the leading 
Soviet protagonist of the idea that following radiation exposure there is 
abnormal afferent activity which leads to some of the well known pathology 
in radiation sickness. This idea would be considered novel by many Western 
investigators and has had few protagonists outside the U.S.S.R. The outlook 
that the CNS play an important role in the production of radiation patho- 
physiology follows directly from the theoretical Soviet position that disease 
is the response of the organism to noxious agents in the environment. The 
CNS mediates such responses and, moreover, attempts to adapt or adjust the 
organism to the disturbing influence. 

The CNS is thought by many Soviets to analyze the internal milieu in 



REVIEW OF RESEARCH IN U.S.S.R. 479 

much the same way it analyzes the physical environment. Pavlov advanced 
such a viewpoint after finding that CRs were possible for introceptive stimuli. 
Much later work, such as that of Bykov (1957), stressed introceptive stimuli 
and introceptive reflexes. Presumably, then, the CNS should analyze the 
complex internal derangements which follow irradiation and adjust the or- 
ganism to them. This view is tenable for pathology which affects known 
introceptors, but is somewhat doubtful in matters such as depression of cell 
mitosis in the gut, testes, or bone marrow, for which no introceptors or 
central regulatory centers are known. 

It has also been thought by some Soviet research workers that nerves 
have a direct controlling influence over mitosis, local regeneration, and 
inflammation. Such mechanisms are usually included under the concept of 
"trophic nerves," which was developed by Botkin, Speransky, and other 
older Russian physiologists. This theory is not completely without support 
in the Western world (Wyburn-Mason, 1950) but a great deal of the evi- 
dence is contradictory. A recent Soviet report for the U.N. (Mastryukova and 
Strzhizhovskii, 1960) summarizes considerable research on the matter and 
concludes that altered levels of epinephrine and adrenal corticoids may be 
the most iinpK)rtant factor in the mitotic depression known to follow various 
insults to the nervous system. 

With this background in mind, it is easier to understand Livanov's view- 
point, though one may not agree with it. In a number of papers Livanov 
(1960) states that radiation with a few hundred r causes abnormal afferent 
inflow, reflected in altered cortical and subcortical encephalograms. There 
follows a complex series of events involving subcortical and cortical inhi- 
bition, leading to altered spinal and autonomic activity, and then gradual 
disinhibition occurs at different times. Livanov states that single doses of 
5-1,000 r commonly cause a stimulatory effect, which may be repeatable, 
but that after total doses of 200-1,000 r there is a general depression of 
EEG activity. EEG reactions to radiation have been described by Livanov 
et al. (1960b) with doses down to 50 r per minute, and such effects are 
said to be predictable with 10-15 r single exposures. 

Grigoryev published a book (1959) which deals with EEG studies on 
humans receiving radiation therapy for tumors of the head or other parts of 
the body. He frequently cites a stimulatory action during the first few 
sessions, then a depressant effect. The precise origin of the claimed changes 
of EEG activity remains obscure, but it will be necessary to rule out stimu- 
lation of the retina, humoral mechanisms, biochemical shifts, and numerous 
other factors before one can conclude with assurance that the EEG change 
indicates an alteration in afferent flow which is communicating information 
about the somatic radiation pathology to the higher nerve centers. 

If significant purposeful, or damaging purposeless, afferent activity oc- 



480 WALTER R. STAHL 

curred after radiation, one might expect that nervous system drugs or CNS 
surgery would radically alter the course of radiation sickness. While many 
agents, including some with actions on the nervous system, can alter radi- 
ation dose-effect curves slightly, there is no evidence that they alter them 
in major ways. It is of basic importance that the most significant delayed 
radiation effects, namely genetic mutations, simulated aging, somatic muta- 
tions, and cancer induction, are all mechanisms in which the organism does 
not adapt to a noxious environmental influence, but suffers innate injury. 
On the other hand, Soviet scientists take a broad view of nervous control 
and may include any neurohumoral and all hormonal mechanisms in the 
category of CNS adaptive responses. Thus, pituitary hormonal changes have 
been scrutinized for importance in radiation sickness. 

The possible role of nerve damage in aging phenomena and in production 
of cancer becomes of special interest from this viewpoint. It is well estab- 
lished that tumors do not have a normal innervation, but it is by no means 
clear that this is of causal significance. Zubareva (1959) reported on the 
detailed histology of nerves in spontaneous breast tumors in mice, and 
Sheveleva (1959) studied nervous influences on the development of a 
Brown-Pearce implanted tumor in rabbits. Similar work is often found in 
the Soviet Bulletin of Experimental Biology aiid Medicine. 

Dzharak'yan (1957) has suggested that postirradiation leucopenia is in 
part due to faulty splenic nerve activity resulting from radiation damage 
to nerve endings. Many studies have dealt with vascular mechanisms in 
irradiated animals and commonly report major alterations in tone following 
several hundred r; this may in part be due to altered levels of circulating 
sympathins, but direct damage to the vessels and their innervation is also 
proposed. Cyclic alterations in the secretory activity of the gut are rep>orted 
by Uspensky (1959) and are said to be due to altered "trophic control" 
predominantly. This will have to be qualified on the basis of known direct 
radiation injury to mucosal cells. 

Many of these studies implicate the autonomic nervous system in radiation 
injury. Soviet authors, especially Lebedinsky, have pointed out that the 
radiation syndrome involves changes in vascular phenomena, thermoregula- 
tion, cardiac activity, respiration, capillary permeability, and numerous 
other autonomic phenomena. He observed changes in hypothalamic stimu- 
lation thresholds for these after irradiation. Studies by others have shown 
changes in the excitability level of the vagal centers after irradiation 
(Gromovskaya, 1959) and similar phenomena. 

Still another aspect of radiation sickness is the specific role of the pitu- 
itary-adrenal axis after irradiation. Results remain contradictory, but some 
Soviet workers feel they have shown a distinct stress reaction to radiation 
injury. Possibly this reaction obtains only in some species and at some doses, 



REVIEW OF RESEARCH IN U.S.S.R. 481 

because much Western work does not support the hypothesis. However, the 
possibiUty of an adrenal cortical mechanism must be kept in mind. 

Alterations in Afferent Activity 

Some stimulatory mechanisms are not pathologic. This sort of reaction 
is typified by the radiation phosphene, covered thoroughly at this conference 
by Lipetz. Recently the Soviet literature also carried a detailed article on 
radiation phosphenes by Gurtovoy and Burdyanskaya (1960), who showed 
this phenomenon in humans receiving 0.2-3 mr. Using blinded subjects, it 
was established that as much as 3 r to the occipital lobe did not produce 
the phosphene, and the evidence is clear that it originates in the retina. 

There are several Soviet reports on electroretinograms. Lebedinsky 
(1959a) cites evidence for changes in frog preparation after 10 r. He has 
discussed alterations in human EEG responses to an increasing light level, 
and it seems entirely possible that subliminal retinal changes are involved 
in the observed alterations. 

Receptors other than the retina may be afTected by radiation. One possi- 
bility is a change in the sensitivity of an undamaged receptor; this seems 
to have been involved in the work of Gorbunova and Rikotova (1958), w^ho 
found that 2 rad of beta rays given locally to the intestinal mucosa led to 
an alteration following standard surface stimulation. Similar findings were 
noted with much higher doses to the skin by Delitsyna (1959a, b), who 
measured afferent activity from the rabbit paw after doses of 500-5,000 r. 
With the larger values, one would expect frank ulceration and changes in 
receptor threshold. 

Other Soviet work suggests that there are changes in afiferent inflow from 
the gut, spleen, bladder, lymph nodes, and elsewhere following irradiation. 
Livanov (1959; Livanov et al., 1960a) has stressed central change due to this 
activity, as noted, and he finds alterations in the firing rates of subcortical 
structures, such as the reticular fomiation and hypothalamus of irradiated 
rabbits, using implanted electrodes. The possibilities of a humoral mech- 
anism and retinal effects need consideration. It does not appear to be clear 
whether radiation may cause changes in the spontaneous firing rates or 
stimulation thresholds for the neurons themselves, as opposed to receptor 
reactions. 

Damage to the Nervous System with Antenatal Irradiation 

Histologic findings after much exposure have been noted. In general, 
production of CNS congenital anomalies has been given some attention in 
the Soviet literature, but not nearly so full a treatment as seen in the 



482 



WALTER R. STAHL 



100 r 



80 



^ 60 



0) 40 



20 



f 



w 



100 r 



80 



<» 60 



40 



20 



^ 



y U U U 



Di 



Q2 



Fig. 2. Effects of 200 r antenatal irradiation (18th day) on the conditional reflex 
stereotype of white rats. Top series are controls: bottom, irradiated; (1), sound (400 
cycles per sec) as a strong positive stimulus; (2), light from small bulb as a weak 
stimulus; (3), sound (800 cycles per sec) as differentiation stimulus. The equalization 
of reactions to stimuli of various strengths and inhibitory after-effect are noted. Re- 
drawn from Piontkovsky et al. (1959). 



works referred to by Hicks, Rugh, and others at this symposium. Piontkov- 
sky and his associates found that CRs in animals antenatally exposed to 
50-200 r showed certain characteristic forms of injury (Fig. 2). 

Figure 3 provides data on a typical stereotype in control and irradiated 
rats. The experimental animal had an equalization of reaction to strong 
and weak stimuli, showed a marked inhibitory after-effect, and displayed 
considerably greater than normal spontaneous motor activity and erratic 
CR learning performance. They often gave a correct response sooner than 



REVIEW OF RESEARCH IN U.S.S.R. 



483 



Radiation EH 



■ Radiation m 



«- Radiation E 



■ Radiation HI 



ooooooooo 
I I — 1 — I — I — I — I 



Radiation I 



Radiation II 






Fig. 3. Similar to figure 1, except that the animal was classified as a weak CNS 
type. Failure of some responses is noted. Redrawn from Khozak ( 1958). 



controls but would not show a fully dependable reflex until many trials 
more than the latter. All these reactions are indicative, in the opinion of 
Soviet scientists, of a failure of internal inhibition. I suggest that the effect 
can be described in other terms as a loss of fine negative feedback or 
inhibitory control. Such a loss would perhaps be expected as the first evi- 
dence of diffuse, mild injury and would account for the pseudo-stimulatory 
action of radiation. 

With in utero irradiation of animals with different CNS types, the specific 
reaction seems to depend in part on this parameter. There is no question 
of injury with these doses, and the animals show major weight loss and 



484 WALTER R. STAHL 

histologic brain atrophy. No Soviet papers demonstrate such changes 
following small antenatal doses. 

The fact that a CR stereotype could be produced at all under these 
conditions raises the question of the sensitivity of the CR method. Pavlov 
noted that the conditional reflex is a basic type of reaction which can occur 
in primitive animals and after extirpation of substantial portions of the 
cortex. After radiation the question is clearly not one of complete failure 
of CR production, but rather of subtle disturbances in higher nervous 
activity which are nearly, but not quite fully, compensated by feedback 
mechanisms. 

Antenatal irradiation is of particular interest in that it makes possible the 
production of nonspecific functional brain damage. The work on CRs sug- 
gests that inhibitory control is damaged particularly, and one might speculate 
that psychopathic personalities also lack normal inhibitory control. Further 
experimentation on emotional or motivational control following antenatal 
irradiation appears promising. 

Conclusion 

It does not seem possible to provide a simple, satisfying summary of the 
multitude of complex experiments discussed. Clearly any definitive statement 
about CNS sensitivity must go into considerable detail concerning the mech- 
anism involved and the form of sensitivity under consideration. 

A final conclusion concerning CNS radiosensitivity must also take cogni- 
zance of certain clinical observations on radiation exposures, namely : pjersons 
receiving even high doses feel no pain and only equivocal mild sensations; 
humans getting doses of 500-1,000 r do not ordinarily show any obvious 
neurologic disturbances, and treatments directed at the nervous system have 
not been helpful in combatting radiation sickness. Many ordinary virus 
infections appear to produce more obvious encephalopathy than radiation, 
and numerous chemicals now used in the environment might evoke CNS 
reactions as great or greater than those seen after radiation exposures at 
moderate levels. 

Not all Soviet radiobiologists place special stress on CNS mechanisms in 
radiation sickness. They point out, however, that the nervous system is a 
major organ system and ought to be given attention in radiation studies, 
during the last two years, there has been a relative decrease in the Soviet 
literature of papers stressing CNS effects, particularly those works citing the 
formal Pavlovian view on the pathophysiology of radiation sickness. 

Following exploration for damage from radiation, certain interesting 
questions of basic biology have now become apparent in connection with 
CNS radiation exposures: 



REVIEW OF RESEARCH IN U.S.S.R. 485 

1. Is radiation damage to the CNS similar to natural aging in this system? 

2. Are the feedback, multiplexing, and other cybernetic mechanisms in the 
CNS such that complete or nearly complete compensations for quite 
massive difTuse neuronal injury can take place? 

3. What tests can be used to bring out difTuse functional damage which is 
well compensated? 

4. Is the role of the nervous system in tumor induction absolutely passive, 
i.e., are the abnormal nervous structures seen in tumors simply the 
result of cells growing out of control? 

5. Does the nervous system exert any direct local control over mitotic ac- 
tivity, even of a limited magnitude, or are cell division and differentia- 
tion controlled wholly by local and circulating biochemical factors? 

6. Is there a complete overlap between the innate mechanisms which are 
tested by the totality of usual behavioral studies and conditional reflex 
techniques? 

7. Does antenatal radiation make it possible to produce functionally dam- 
aged nervous systems comparable to those found in psychopathic or 
otherwise mentally disturbed humans? 

8. What is the role of direct autonomic system damage in such proven 
postirradiation eflfects as vascular reactivity changes? 

These questions difTer considerably from the problem of central nervous 
radiosensitivity as posed originally and emphasize the continuing need for 
radiological research at the basic scientific level. 

References 

Aleksandrovskaya, M. M. 1958. Certain data on the effects of ionizing radiation on 

the morphology of the central nervous system in animals. Trudy Inst. Vysh. Nerv. 

Deyatel. 4, 221-224. 
Alekseyeva, G. I. 1958. The condition of the receptor apparatus in the walls of large 

vessels following roentgen irradiation. Byull. Eksptl. Biol. Med. 46, No. 10, 122-124. 
Anisomova-AIeksandrova, V. V. 1959. Morphological changes in various components 

of the peripheral ner\'ous system after exposure of the organism to ionizing radia- 
tion. Med. Radiol. 4, No. 11, 3-9. 
Bibikova, A. F. 1959. Demyelization of central nervous system fibres following total 

body ionizing radiation of animals. Arkhiv Patol. 21, No. 5, 19-25. 
Biryukov, D. A. 1957. Data on the problem of the effect of penetrating radiation on 

central (nervous) inhibition. In "Problemy Fiziologii Tsentral'noy Nervnoy Sis- 

temy," pp. 73-83. Medgiz, Moscow and Leningrad. 
Brazier, M. A. B., ed. 1959. "The Central Ner\'ous System and Behavior," Conference. 

Josiah Macy, Jr., Foundation, New York. 
Brodskaya, Ye. A., and Merkulova, I. P. 1956. Concerning late changes in the brain 

following roentgen irradiation. Vestnik. Roentgenol, i Radiol. 31, No. 2, 7-13. 
Brodskaya, Ye. A. 1958. Reflex effects from internal organs on the functions of the 

gastrointestinal tract in irradiated animals. Arkhiv Patol. 20, No. 11, 53-58. 



486 WALTER R. STAHL 

Bykov, K. M. 1957. "The Cerebral Cortex and the Internal Organs" (translated by 
W. H. Gantt). Chemical Publ. Co., New York. 

Cherkasov, V. F. 1960. A review of works on the influence of ionizing radiation on 
the nervous system. Med. Radiol. 5, No. 2, 93-96. 

Delitsyna, N. S. 1959a. Investigation of receptor function in irradiated portions of the 
body using experimental animals. Med. Radiol. 4, No. 8, 17-20. 

Delitsyna, N. S. 1959b. Concerning some changes in receptor systems under the in- 
fluence of x-rays. Works of the All-Union Conf. on Medical Radiology, Exptl. Med. 
Radiol., AEC-tr-3661 (book 1), U.S. Atomic Energy Commission AEC-51-60. 

Dzharak'yan, T. K. 1957. Proc. All-Union Conf. on Med. Radiol., pp. 44-51, ab- 
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